Inhibitors of Human EZH2, and Methods of Use Thereof

ABSTRACT

The invention relates to inhibition of wild-type and certain mutant forms of human histone methyltransferase EZH2, the catalytic subunit of the PRC2 complex which catalyzes the mono- through tri-methylation of lysine 27 on histone H3 (H3-K27). In one embodiment the inhibition is selective for the mutant form of the EZH2, such that trimethylation of H3-K27, which is associated with certain cancers, is inhibited. The methods can be used to treat cancers including follicular lymphoma and diffuse large B-cell lymphoma (DLBCL). Also provided are methods for identifying small molecule selective inhibitors of the mutant forms of EZH2 and also methods for determining responsiveness to an EZH2 inhibitor in a subject.

RELATED REFERENCE

This application is a continuation-in-part of the U.S. patentapplication Ser. No. 13/230,703, filed Sep. 12, 2011, which claimspriority to, and the benefit of, U.S. Ser. No. 61/381,684, filed Sep.10, 2010, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to inhibition of wild-type and certain mutantforms of human histone methyltransferase EZH2, the catalytic subunit ofthe PRC2 complex which catalyzes the mono- through tri-methylation oflysine 27 on histone H3 (H3-K27), methods for treating cancers includingfollicular lymphoma and diffuse large B-cell lymphoma (DLBCL) andmethods for determining responsiveness to an EZH2 inhibitor in asubject.

BACKGROUND

In eukaryotic cells DNA is packaged with histones to form chromatin.Approximately 150 base pairs of DNA are wrapped twice around an octamerof histones (two each of histones 2A, 2B, 3 and 4) to form a nucleosome,the basic unit of chromatin. Changes in the ordered structure ofchromatin can lead to alterations in transcription of associated genes.This process is highly controlled because changes in gene expressionpatterns can profoundly affect fundamental cellular processes, such asdifferentiation, proliferation and apoptosis. Control of changes inchromatin structure (and hence of transcription) is mediated by covalentmodifications to histones, most notably of their N-terminal tails. Thesemodifications are often referred to as epigenetic because they can leadto heritable changes in gene expression, but they do not affect thesequence of the DNA itself. Covalent modifications (for example,methylation, acetylation, phosphorylation and ubiquitination) of theside chains of amino acids are enzymatically mediated.

The selective addition of methyl groups to specific amino acid sites onhistones is controlled by the action of a unique family of enzymes knownas histone methyltransferases (HMTs). The level of expression of aparticular gene is influenced by the presence or absence of one or moremethyl groups at a relevant histone site. The specific effect of amethyl group at a particular histone site persists until the methylgroup is removed by a histone demethylase, or until the modified histoneis replaced through nucleosome turnover. In a like manner, other enzymeclasses can decorate DNA and histones with other chemical species, andstill other enzymes can remove these species to provide control of geneexpression.

The orchestrated collection of biochemical systems behindtranscriptional regulation must be tightly controlled in order for cellgrowth and differentiation to proceed optimally. Disease states resultwhen these controls are disrupted by aberrant expression and/or activityof the enzymes responsible for DNA and histone modification. In humancancers, for example, there is a growing body of evidence to suggestthat dysregulated epigenetic enzyme activity contributes to theuncontrolled cell proliferation associated with cancer as well as othercancer-relevant phenotypes such as enhanced cell migration and invasion.Beyond cancer, there is growing evidence for a role of epigeneticenzymes in a number of other human diseases, including metabolicdiseases (such as diabetes), inflammatory diseases (such as Crohn'sdisease), neurodegenerative diseases (such as Alzheimer's disease), andcardiovascular diseases. Therefore, selectively modulating the aberrantaction of epigenetic enzymes holds great promise for the treatment of arange of diseases.

Histone Methyltransferase EZH2

Polycomb group (PcG) and trithorax group (trxG) proteins are known to bepart of the cellular memory system. Francis et al. (2001) Nat Rev MolCell Biol 2:409-21; Simon et al. (2002) Curr Opin Genet Dev 12:210-8.Both groups of proteins are involved in maintaining the spatial patternsof homeotic box (Hox) gene expression, which are established early inembryonic development by transiently expressed segmentation genes. Ingeneral, PcG proteins are transcriptional repressors that maintain the“off state,” and trxG proteins are transcriptional activators thatmaintain the “on state.” Because members of PcG and trxG proteinscontain intrinsic histone methyltransferase (HMTase) activity, PcG andtrxG proteins may participate in cellular memory through methylation ofcore histones. Beisel et al. (2002) Nature 419:857-62; Cao et al. (2002)Science 298:1039-43; Czermin et al. (2002) Cell 111:185-96; Kuzmichev etal. (2002) Genes Dev 16:2893-905; Milne et al. (2002) Mol Cell10:1107-17; Muller et al. (2002) Cell 111:197-208; Nakamura et al.(2002) Mol Cell 10:1119-28.

Biochemical and genetic studies have provided evidence that DrosophilaPcG proteins function in at least two distinct protein complexes, thePolycomb repressive complex 1 (PRC1) and the ESC-E(Z) complex (alsoknown as Polycomb repressive complex 2 (PRC2)), although thecompositions of the complexes may be dynamic. Otte et al. (2003) CurrOpin Genet Dev 13:448-54. Studies in Drosophila (Czermin et al. (supra);Muller et al. (supra)) and mammalian cells (Cao et al. (supra);Kuzmichev et al. (supra)) have demonstrated that the ESC-E(Z)/EED-EZH2(i.e., PRC2) complexes have intrinsic histone methyltransferaseactivity. Although the compositions of the complexes isolated bydifferent groups are slightly different, they generally contain EED,EZH2, SUZ12, and RbAp48 or Drosophila homologs thereof. However, areconstituted complex comprising only EED, EZH2, and SUZ12 retainshistone methyltransferase activity for lysine 27 of histone H3. U.S.Pat. No. 7,563,589 (incorporated by reference).

Of the various proteins making up PRC2 complexes, EZH2 (Enhancer ofZeste Homolog 2) is the catalytic subunit. The catalytic site of EZH2 inturn is present within a SET domain, a highly conserved sequence motif(named after Su(var)3-9, Enhancer of Zeste, Trithorax) that is found inseveral chromatin-associated proteins, including members of both theTrithorax group and Polycomb group. SET domain is characteristic of allknown histone lysine methyltransferases except the H3-K79methyltransferase DOT1.

In addition to Hox gene silencing, PRC2-mediated histone H3-K27methylation has been shown to participate in X-inactivation. Plath etal. (2003) Science 300:131-5; Silva et al. (2003) Dev Cell 4:481-95.Recruitment of the PRC2 complex to Xi and subsequent trimethylation onhistone H3-K27 occurs during the initiation stage of X-inactivation andis dependent on Xist RNA. Furthermore, EZH2 and its associated histoneH3-K27 methyltransferase activity was found to mark differentially thepluripotent epiblast cells and the differentiated trophectoderm. Erhardtet al. (2003) Development 130:4235-48).

Consistent with a role of EZH2 in maintaining the epigeneticmodification patterns of pluripotent epiblast cells, Cre-mediateddeletion of EZH2 results in loss of histone H3-K27 methylation in thecells. Erhardt et al. (supra). Further, studies in prostate and breastcancer cell lines and tissues have revealed a strong correlation betweenthe levels of EZH2 and SUZ12 and the invasiveness of these cancers(Bracken et al. (2003) EMBO J 22:5323-35; Kirmizis et al. (2003) MolCancer Ther 2:113-21; Kleer et al. (2003) Proc Natl Acad Sci USA100:11606-11; Varambally et al. (2002) Nature 419:624-9), indicatingthat dysfunction of the PRC2 complex may contribute to cancer.

Recently, somatic mutations of EZH2 were reported to be associated withfollicular lymphoma (FL) and the germinal center B cell-like (GCB)subtype of diffuse large B-cell lymphoma (DLBCL). Morin et al. (2010)Nat Genet 42:181-5. In all cases, occurrence of the mutant EZH2 gene wasfound to be heterozygous, and expression of both wild-type and mutantalleles was detected in the mutant samples profiled by transcriptomesequencing. Currently, the standard of care for the treatment of mostcases of DLBCL is the R—CHOP regimen. However, the outcome of thisregimen is far from satisfactory. Therefore, there is a great medicalneed to identify novel and effective therapies, optionally based on thegenetic profiles of the subject.

SUMMARY OF THE INVENTION

The invention is based upon the discovery that cells expressing certainEZH2 mutants are more responsive to EZH2 inhibitors than cellsexpressing wild type EZH2.

The invention features a method for treating or alleviating a symptom ofcancer or precancerous condition in a subject administering to a subjectexpressing a mutant EZH2 comprising a mutation in the substrate pocketdomain as defined in SEQ ID NO: 6 a therapeutically effective amount ofan EZH2 inhibitor.

The invention also features a method of determining a responsiveness ofa subject having a cancer or a precancerous condition to an EZH2inhibitor by providing a sample from the subject; and detecting amutation in the EZH2 substrate pocket domain as defined in SEQ ID NO: 6;and the presence of said mutation indicates the subject is responsive tothe EZH2 inhibitor.

The mutant EZH2 of the present invention is a mutant EZH2 polypeptide ora nucleic acid sequence encoding a mutant EZH2 polypeptide. Preferably,the mutant EZH2 comprises a mutation at amino acid position 677, 687,674, 685, or 641 of SEQ ID NO: 1. More preferably, mutation is selectedfrom the group consisting of a substitution of glycine (G) for the wildtype residue alanine (A) at amino acid position 677 of SEQ ID NO: 1(A677G); a substitution of valine (V) for the wild type residue alanine(A) at amino acid position 687 of SEQ ID NO: 1 (A687V); a substitutionof methionine (M) for the wild type residue valine (V) at amino acidposition 674 of SEQ ID NO: 1 (V674M); a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 685 of SEQID NO: 1 (R685H); a substitution of cysteine (C) for the wild typeresidue arginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685C);a substitution of phenylalanine (F) for the wild type residue tyrosine(Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F); a substitutionof histidine (H) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641H); a substitution of asparagine (N)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641N); a substitution of serine (S) for the wild type residuetyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641S); and asubstitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641C).

The subject of the present invention includes any human subject who hasbeen diagnosed with, has symptoms of, or is at risk of developing acancer or a precancerous condition. For example, the cancer is lymphoma,leukemia or melanoma. Preferably, the lymphoma is non-Hodgkin lymphoma,follicular lymphoma or diffuse large B-cell lymphoma. Alternatively, theleukemia is chronic myelogenous leukemia (CML). The precancerouscondition is myelodysplastic syndromes (MDS, formerly known aspreleukemia).

Preferred EZH2 inhibitor for the methods of the present invention isselected from compounds listed in Table 1.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is two graphs establishing that B-cell lymphoma-associatedmutants of EZH2 are active histone methyltransferases. In vitromethyltransferase activity of PRC2 complexes containing wild-type andvarious Y641 mutants of EZH2 was measured as (A) methyl transferreactions using a peptide (H3 21-44) as substrate, and (B) methyltransfer reactions using avian nucleosomes as substrate. Symbols:wild-type (), Y641F (∘), Y641H (□), Y641N (▪), and Y641S (▴). CPM iscounts per minute, referring to scintillation counting as a result of ³Hradiation.

FIG. 2 is four graphs establishing that PRC2 complexes containing mutantEZH2 preferentially catalyze di- and tri-methylation of histone H3-K27.(A) Methyltransferase activity of mutant and wild-type (WT) complexes onunmethylated peptide (open bars), monomethylated peptide (hashed bars),and dimethylated peptide (closed bars). (B) Affinity for peptidesubstrates as judged by K_(1/2) is similar across all peptidemethylation states for PRC2 complexes containing wild-type (∘), Y641F(), Y641H (□), Y641N (▪), and Y641S (▴) EZH2. Note that the variationin K_(1/2) values across all substrates and all enzyme forms is lessthan 3.5-fold. For any particular methylation state of substrate thevariation in K_(1/2) value is less than 2-fold. (C) Enzyme turnovernumber (k_(cat)) varies with substrate methylation status in opposingways for WT and Y641 mutants of EZH2. The k_(cat) decreases withincreasing K27 methylation states for wild-type (∘), but increases forY641F (), Y641H (□), Y641N (▪), and Y641S (▴) mutants of EZH2. (D)Catalytic efficiency (k_(cat)/K_(1/2)) decreases with increasing K27methylation states for wild-type (∘), but increases for Y641F (), Y641H(□), Y641N (▪), and Y641S (▴) mutants of EZH2. In panels B-D, the linesdrawn to connect the data points are not intended to imply anymathematical relationship; rather, they are merely intended to serve asvisual aides.

FIG. 3A is a trio of graphs depicting predicted relative levels ofH3-K27me3 (top panel), H3-K27me2 (middle panel), and H3-K27me1 (bottompanel) for cells containing different EZH2 mutants. Simulations wereperformed using a coupled enzyme steady state velocity equation and thesteady state kinetic parameters shown in Table 2. All values arerelative to the homozygous WT EZH2-containing cells and assumesaturating concentrations of intracellular SAM, relative to Km andintracellular nucleosome concentrations similar to Km.

FIG. 3B is a series of Western blot analyses of relative patterns ofH3-K27 methylation status for lymphoma cell lines homozygous for WTEZH2, or heterozygous for the indicated EZH2 Y641 mutation. Panels fromtop to bottom depict the results of probing with antibodies specific forthe following: total EZH2; H3-K27me3; H3-K27me2; H3-K27me1; and totalhistone H3 as loading control.

FIG. 4 depicts selected proposed mechanisms leading to aberrantly highlevels of trimethylation on histone H3-K27 in cancer. These include: a)mutation of Y641 in EZH2 resulting in a change in substrate preferencefrom the nonmethylated to the mono- and di-methylated histone H3-K27; b)overexpression of EZH2; c) mutations in UTX that inactivate enzymefunction, causing a decrease in demethylation of H3-K27me3; and d)overexpression of the PRC2 complex subunit PHF19/PCL3 that leads toincreases in recruitment of the PRC2 complex to specific genes and anincrease in histone H3-K27 trimethylation. In all four models thealteration leads to aberrant histone H3-K27 trimethylation in theproximal promoter regions of genes resulting in transcriptionalrepression of key genes in cancer.

FIG. 5 depicts a SDS-PAGE gel showing that the expression levels of eachof the five-component PRC2 complexes are similar with mutant andwild-type EZH2.

FIG. 6 is a pair of tables showing that mutant and wild-type (WT) PRC2complexes display strong substrate preference for H3-K27-containingpeptides. Each enzyme was tested against a panel of overlapping 15-merpeptides covering all of H3 and H4. Activity was measured as velocity(CPM per minute), and the reported value represents the mean of twoindependent determinations for each reaction. For all the complexes themost favored peptide was H3:16-30. WT complex had greater than 6-foldmore activity against this peptide than any of the mutant complexes.

FIG. 7 is a graph depicting inhibitory potency ofS-adenosyl-L-homocysteine (SAH) against EZH2 WT and Y641 mutants ofEZH2. The X axis shows log concentration of SAH; theY axis shows percentinhibition.

FIG. 8 is a graph depicting inhibitory potency of Compound 75 againstEZH2 WT and Y641 mutants of EZH2. The X axis shows log concentration ofCompound 75; the Y axis shows percent inhibition.

FIG. 9 depicts a Western Blot analysis of relative levels of H3-K27me1,me2 and me3 in a cell line pane, including multiple DLBCL linesexpressing WT or Y641 mutatnt EZH2. Histones were extracted from thecell lines shown, fractionated by SDS-PAGE on a 4-20% gel, transferredto nitrocellulose membranes, and probed with antibodies to Histone H3,H3-K27me1, me2, or me3.

FIG. 10 depicts an immunocytochemistry analysis of H3 and H3-K27me3levels in a panel of WT and Y641 mutant lymphoma cell lines. Cellpellets from the indicated cell lines were fixed and embedded inparaffin. Slides were prepared and levels of H3 and H3-K27me3 wereevaluated by immunocytochemistry using antibodies to histone H3, orH3-K27me3.

FIG. 11 depicts an immunocytochemistry analysis of H3 and H3-K27me2levels in a panel of WT and Y641 mutant lymphoma cell lines. Cellpellets from the indicated cell lines were fixed and embedded inparaffin. Slides were prepared and levels of H3 and H3-K27me2 wereevaluated by immunocytochemistry using antibodies to histone H3, orH3-K27me2.

FIG. 12 is a graph depicting the inhibition of global H3-K27me3 levelsby EZH2 inhibitor treatment in Y641 mutant WSU-DLCL2 cells. WSU-DLCL2cells were treated for 4 days with the indicated concentrations of EZH2inhibitor A or B. Following compound treatment, histones were extracted,fractionated by SDS-PAGE on a 4-20% gel, transferred to nitrocellulosemembranes, and probed with antibodies to Histone H3, or H3-K27me3.

FIG. 13 is a graph showing that the EZH2 inhibitors can blockproliferation of a Y641 mutant WSU-DLCL2 cells, but has little effect onnon Y641 mutant OCI-LY19 cells. Cells were incubated in the presence ofincreasing concentrations of EZH2 inhibitor A or B for eleven days.Vehicle treated (DMSO) cells were included as controls. Cell number andviability was determined using the Guava Viacount assay in a GuavaEasyCyte Plus instrument. Cells were split and media and compound wasreplenished every 3-4 days.

FIG. 14 is a graph showing the presence of an EZH2 (Y641) mutationand/or high H3-K27me3 and low H3-K27me2 levels predict sensitivity toEZH2 inhibitors. Cell lines were maintained in the presence ofincreasing concentrations of one EZH2 inhibitor up to 25 μM. Viablecells counts were used to derive IC₉₀ values after 11 days of treatment.Results are plotted with cell lines segregated according to EZH2mutational status (A), or segregated according to H3-K27me2 andH3-K27me3 levels (B). In both plots, the line shows the average IC90values from the indicated cell line group.

FIG. 15 is a panel of graphs showing velocity vs. enzyme concentrationmeasured for wild-type, A677G or A687V EZH2. Biotinylated peptidesrepresenting histone H3 residues 21-44 containing un-, mono-, di- ortrimethyl lysine 27 (H3K27me0 through H3K27me3) were assayed at a fixedconcentration with a dilution series of the indicated EZH2 enzyme.Timepoints were sampled over the course of 90 minutes and 3H-SAMincorporation at lysine 27 of the H3 peptide was measured by capturingthe peptide in a Flashplate and reading the counts per minute (CPM).Linear regression of the timecourse yielded enzyme velocity in CPM perminute (CPM/min) which was plotted as a function of enzymeconcentration.

DETAILED DESCRIPTION

Chromatin structure is important in gene regulation and epigeneticinheritance. Post-translational modifications of histones, such asmethylation are involved in the establishment and maintenance ofhigher-order chromatin structure.

EZH2 Mutants

EZH2 is a histone methyltransferase that is the catalytic subunit of thePRC2 complex which catalyzes the mono- through tri-methylation of lysine27 on histone H3 (H3-K27).

Point mutations of the EZH2 gene at a single amino acid residue (e.g.,Tyr641, herein referred to as Y641) of EZH2 have been reported to belinked to subsets of human B-cell lymphoma. Morin et al. (2010) NatGenet 42(2):181-5. In particular, Morin et al. reported that somaticmutations of tyrosine 641 (Y641F, Y641H, Y641N, and Y641S) of EZH2 wereassociated with follicular lymphoma (FL) and the germinal center Bcell-like (GCB) subtype of diffuse large B-cell lymphoma (DLBCL). Themutant allele is always found associated with a wild-type allele(heterozygous) in disease cells, and the mutations were reported toablate the enzymatic activity of the PRC2 complex for methylating anunmodified peptide substrate.

The present invention is based in part upon the surprising discoverythat cells expressing an EZH2 mutant are more sensitive to EZH2inhibitors of the instant invention than cells expressing wild typeEZH2. Accordingly, an aspect of the present invention relates to methodsfor treating or alleviating a symptom of cancer or precancerouscondition in a subject by administering to a subject expressing a mutantEZH2 a therapeutically effective amount of an EZH2 inhibitor. The mutantEZH2 of the present invention refers to a mutant EZH2 polypeptide or anucleic acid sequence encoding a mutant EZH2 polypeptide. Preferably themutant EZH2 comprises one or more mutations in its substrate pocketdomain as defined in SEQ ID NO: 6. For example, the mutation may be asubstitution, a point mutation, a nonsense mutation, a miss sensemutation, a deletion, or an insertion. Exemplary substitution amino acidmutation includes a substitution at amino acid position 677, 687, 674,685, or 641 of SEQ ID NO: 1, such as, but is not limited to asubstitution of glycine (G) for the wild type residue alanine (A) atamino acid position 677 of SEQ ID NO: 1 (A677G); a substitution ofvaline (V) for the wild type residue alanine (A) at amino acid position687 of SEQ ID NO: 1 (A687V); a substitution of methionine (M) for thewild type residue valine (V) at amino acid position 674 of SEQ ID NO: 1(V674M); a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685H); asubstitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 685 of SEQ ID NO: 1 (R685C); a substitution ofphenylalanine (F) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641F); a substitution of histidine (H)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641H); a substitution of asparagine (N) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641N);a substitution of serine (S) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641S); or a substitution ofcysteine (C) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641C).

The mutation of the present invention may also include a substitution ofserine (S) for the wild type residue asparagine (N) at amino acidposition 322 of SEQ ID NO: 3 (N322S), a substitution of glutamine (Q)for the wild type residue arginine (R) at amino acid position 288 of SEQID NO: 3 (R288Q), a substitution of isoleucine (I) for the wild typeresidue threonine (T) at amino acid position 573 of SEQ ID NO: 3(T573I), a substitution of glutamic acid (E) for the wild type residueaspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 (D664E), asubstitution of glutamine (Q) for the wild type residue arginine (R) atamino acid position 458 of SEQ ID NO: 5 (R458Q), a substitution oflysine (K) for the wild type residue glutamic acid (E) at amino acidposition 249 of SEQ ID NO: 3 (E249K), a substitution of cysteine (C) forthe wild type residue arginine (R) at amino acid position 684 of SEQ IDNO: 3 (R684C), a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 628 of SEQ ID NO: 21 (R628H), asubstitution of histidine (H) for the wild type residue glutamine (Q) atamino acid position 501 of SEQ ID NO: 5 (Q501H), a substitution ofasparagine (N) for the wild type residue aspartic acid (D) at amino acidposition 192 of SEQ ID NO: 3 (D192N), a substitution of valine (V) forthe wild type residue aspartic acid (D) at amino acid position 664 ofSEQ ID NO: 3 (D664V), a substitution of leucine (L) for the wild typeresidue valine (V) at amino acid position 704 of SEQ ID NO: 3 (V704L), asubstitution of serine (S) for the wild type residue proline (P) atamino acid position 132 of SEQ ID NO: 3 (P132S), a substitution oflysine (K) for the wild type residue glutamic acid (E) at amino acidposition 669 of SEQ ID NO: 21 (E669K), a substitution of threonine (T)for the wild type residue alanine (A) at amino acid position 255 of SEQID NO: 3 (A255T), a substitution of valine (V) for the wild type residueglutamic acid (E) at amino acid position 726 of SEQ ID NO: 3 (E726V), asubstitution of tyrosine (Y) for the wild type residue cysteine (C) atamino acid position 571 of SEQ ID NO: 3 (C571Y), a substitution ofcysteine (C) for the wild type residue phenylalanine (F) at amino acidposition 145 of SEQ ID NO: 3 (F145C), a substitution of threonine (T)for the wild type residue asparagine (N) at amino acid position 693 ofSEQ ID NO: 3 (N693T), a substitution of serine (S) for the wild typeresidue phenylalanine (F) at amino acid position 145 of SEQ ID NO: 3(F145S), a substitution of histidine (H) for the wild type residueglutamine (Q) at amino acid position 109 of SEQ ID NO: 21 (Q109H), asubstitution of cysteine (C) for the wild type residue phenylalanine (F)at amino acid position 622 of SEQ ID NO: 21 (F622C), a substitution ofarginine (R) for the wild type residue glycine (G) at amino acidposition 135 of SEQ ID NO: 3 (G135R), a substitution of glutamine (Q)for the wild type residue arginine (R) at amino acid position 168 of SEQID NO: 5 (R168Q), a substitution of arginine (R) for the wild typeresidue glycine (G) at amino acid position 159 of SEQ ID NO: 3 (G159R),a substitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 310 of SEQ ID NO: 5 (R310C), a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 561 of SEQ ID NO: 3 (R561H), a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 634 of SEQID NO: 21 (R634H), a substitution of arginine (R) for the wild typeresidue glycine (G) at amino acid position 660 of SEQ ID NO: 3 (G660R),a substitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 181 of SEQ ID NO: 3 (Y181C), a substitution ofarginine (R) for the wild type residue histidine (H) at amino acidposition 297 of SEQ ID NO: 3 (H297R), a substitution of serine (S) forthe wild type residue cysteine (C) at amino acid position 612 of SEQ IDNO: 21 (C612S), a substitution of tyrosine (Y) for the wild type residuehistidine (H) at amino acid position 694 of SEQ ID NO: 3 (H694Y), asubstitution of alanine (A) for the wild type residue aspartic acid (D)at amino acid position 664 of SEQ ID NO: 3 (D664A), a substitution ofthreonine (T) for the wild type residue isoleucine (I) at amino acidposition 150 of SEQ ID NO: 3 (1150T), a substitution of arginine (R) forthe wild type residue isoleucine (I) at amino acid position 264 of SEQID NO: 3 (1264R), a substitution of leucine (L) for the wild typeresidue proline (P) at amino acid position 636 of SEQ ID NO: 3 (P636L),a substitution of threonine (T) for the wild type residue isoleucine (I)at amino acid position 713 of SEQ ID NO: 3 (1713T), a substitution ofproline (P) for the wild type residue glutamine (Q) at amino acidposition 501 of SEQ ID NO: 5 (Q501P), a substitution of glutamine (Q)for the wild type residue lysine (K) at amino acid position 243 of SEQID NO: 3 (K243Q), a substitution of aspartic acid (D) for the wild typeresidue glutamic acid (E) at amino acid position 130 of SEQ ID NO: 5(E130D), a substitution of glycine (G) for the wild type residuearginine (R) at amino acid position 509 of SEQ ID NO: 3 (R509G), asubstitution of histidine (H) for the wild type residue arginine (R) atamino acid position 566 of SEQ ID NO: 3 (R566H), a substitution ofhistidine (H) for the wild type residue aspartic acid (D) at amino acidposition 677 of SEQ ID NO: 3 (D677H), a substitution of asparagine (N)for the wild type residue lysine (K) at amino acid position 466 of SEQID NO: 5 (K466N), a substitution of histidine (H) for the wild typeresidue arginine (R) at amino acid position 78 of SEQ ID NO: 3 (R78H), asubstitution of methionine (M) for the wild type residue lysine (K) atamino acid position 1 of SEQ ID NO: 6 (K6M), a substitution of leucine(L) for the wild type residue serine (S) at amino acid position 538 ofSEQ ID NO: 3 (S538L), a substitution of glutamine (Q) for the wild typeresidue leucine (L) at amino acid position 149 of SEQ ID NO: 3 (L149Q),a substitution of valine (V) for the wild type residue leucine (L) atamino acid position 252 of SEQ ID NO: 3 (L252V), a substitution ofvaline (V) for the wild type residue leucine (L) at amino acid position674 of SEQ ID NO: 3 (L674V), a substitution of valine (V) for the wildtype residue alanine (A) at amino acid position 656 of SEQ ID NO: 3(A656V), a substitution of aspartic acid (D) for the wild type residuealanine (A) at amino acid position 731 of SEQ ID NO: 3 (Y731D), asubstitution of threonine (T) for the wild type residue alanine (A) atamino acid position 345 of SEQ ID NO: 3 (A345T), a substitution ofaspartic acid (D) for the wild type residue alanine (A) at amino acidposition 244 of SEQ ID NO: 3 (Y244D), a substitution of tryptophan (W)for the wild type residue cysteine (C) at amino acid position 576 of SEQID NO: 3 (C576W), a substitution of lysine (K) for the wild type residueasparagine (N) at amino acid position 640 of SEQ ID NO: 3 (N640K), asubstitution of lysine (K) for the wild type residue asparagine (N) atamino acid position 675 of SEQ ID NO: 3 (N675K), a substitution oftyrosine (Y) for the wild type residue aspartic acid (D) at amino acidposition 579 of SEQ ID NO: 21 (D579Y), a substitution of isoleucine (I)for the wild type residue asparagine (N) at amino acid position 693 ofSEQ ID NO: 3 (N693I), and a substitution of lysine (K) for the wild typeresidue asparagine (N) at amino acid position 693 of SEQ ID NO: 3(N693K).

The mutation of the present invention may be a frameshift at amino acidposition 730, 391, 461, 441, 235, 254, 564, 662, 715, 405, 685, 64, 73,656, 718, 374, 592, 505, 730, or 363 of SEQ ID NO: 3, 5 or 21 or thecorresponding nucleotide position of the nucleic acid sequence encodingSEQ ID NO: 3, 5, or 21. The mutation of the EZH2 may also be aninsertion of a glutamic acid (E) between amino acid positions 148 and149 of SEQ ID NO: 3, 5 or 21. Another example of EZH2 mutation is adeletion of glutamic acid (E) and leucine (L) at amino acid positions148 and 149 of SEQ ID NO: 3, 5 or 21. The mutant EZH2 may furthercomprise a nonsense mutation at amino acid position 733, 25, 317, 62,553, 328, 58, 207, 123, 63, 137, or 60 of SEQ ID NO: 3, 5 or 21.

It has also been unexpectedly discovered that the wild-type (WT) EZH2enzyme displays greatest catalytic efficiency (kcat/K) for the zero- tomono-methylation reaction of H3-K27 and lesser efficiency for subsequent(mono- to di- and di- to tri-methylation) reactions; whereas, in starkcontrast, the disease-associated Y641 mutations display very limitedability to perform the first methylation reaction but have enhancedcatalytic efficiency for the subsequent reactions relative to wild-typeenzyme. These results imply that the malignant phenotype of diseaseexploits the combined activities of a H3-K27 mono-methylating enzyme(PRC2 containing WT EZH2 or EZH1) together with PRC2 containing mutantEZH2 for augmented conversion of H3-K27 to the tri-methylated form(H3-K27me3). Therefore, an aspect of the present invention relates toinhibiting the activity of EZH2, including certain mutant forms of EZH2.In one embodiment the present invention relates to inhibitingselectively the activity of certain mutant forms of EZH2.

While not intending to be bound by any one theory, it is hypothesizedthat the mutation of EZH2 in its substrate pocket domain may facilitatemultiple rounds of H3-K27 methylation by impacting the H-bonding patternand/or steric crowding in the active site of the enzyme-bisubstrateternary complex, affecting the formation of a proper water channel fordeprotonation of the reacting lysine.

Human EZH2 nucleic acids and polypeptides have previously beendescribed. See, e.g., Chen et al. (1996) Genomics 38:30-7 [746 aminoacids]; Swiss-Prot Accession No. Q15910 [746 amino acids]; GenBankAccession Nos. NM_(—)004456 and NP_(—)004447 (isoform a [751 aminoacids]); and GenBank Accession Nos. NM_(—)152998 and NP_(—)694543(isoform b [707 amino acids]), each of which is incorporated herein byreference in its entirety.

Amino acid sequence of human EZH2 (Swiss-Prot Accession No. Q15910)(SEQ ID NO: 1)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPmRNA sequence of human EZH2, transcript variant 1 (GenBankAccession No. NM_004456) (SEQ ID NO: 2)ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgtttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccggcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcgggacgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcggaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctgatgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatcttaaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctcattgcgcgggactagggagtgttcggtgaccagtgacttggattttccaacacaagtcatcccattaaagactctgaatgcagttgcttcagtacccataatgtattcttggtctcccctacagcagaattttatggtggaagatgaaactgttttacataacattccttatatgggagatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacctcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgcacttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcctacatcgtaagtgcaattattcttttcatgcaacacccaacacttataagcggaagaacacagaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagccttgtgacagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaaccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtgcccgtgctacctggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgctgaccattgggacagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaaaaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtccttgttgaatcFull amino acid of EZH2, isoform a (GenBank Accession No. NP_004447)(SEQ ID NO: 3)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHRKCNYSFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPmRNA sequence of human EZH2, transcript variant 2 (GenBankAccession No. NM_152998) (SEQ ID NO: 4)ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgtttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccggcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcgggacgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcggaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctgatgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatcttaaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctcattgcgcgggactagggaggtggaagatgaaactgttttacataacattccttatatgggagatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacctcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgcacttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcctacatccttttcatgcaacacccaacacttataagcggaagaacacagaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagcCttgtgacagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaaccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtgcccgtgctacctggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgctgaccattgggacagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaaaaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtc cttgttgaatcFull amino acid of EZH2, isoform b (GenBank Accession No. NP_694543)(SEQ ID NO: 5) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTREVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGI EREMEIPFull amino acid of EZH2, isoform e (GenBank Accession No.NP_001190178.1) (SEQ ID NO: 21)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSCSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKGQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPHomo sapiens enhancer of zeste homolog 2 (Drosophila) (EZH2), transcriptvariant 5, mRNA (GenBank Accession No. NM_001203249.1) (SEQ ID NO: 22)GACGACGTTCGCGGCGGGGAACTCGGAGTAGCTTCGCCTCTGACGTTTCCCCACGACGCACCCCGAAATCCCCCTGAGCTCCGGCGGTCGCGGGCTGCCCTCGCCGCCTGGTCTGGCTTTATGCTAAGTTTGAGGGAAGAGTCGAGCTGCTCTGCTCTCTATTGATTGTGTTTCTGGAGGGCGTCCTGTTGAATTCCCACTTCATTGTGTACATCCCCTTCCGTTCCCCCCAAAAATCTGTGCCACAGGGTTACTTTTTGAAAGCGGGAGGAATCGAGAAGCACGATCTTTTGGAAAACTTGGTGAACGCCTAAATAATCATGGGCCAGACTGGGAAGAAATCTGAGAAGGGACCAGTTTGTTGGCGGAAGCGTGTAAAATCAGAGTACATGCGACTGAGACAGCTCAAGAGGTTCAGACGAGCTGATGAAGTAAAGAGTATGTTTAGTTCCAATCGTCAGAAAATTTTGGAAAGAACGGAAATCTTAAACCAAGAATGGAAACAGCGAAGGATACAGCCTGTGCACATCCTGACTTCTTGTTCGGTGACCAGTGACTTGGATTTTCCAACACAAGTCATCCCATTAAAGACTCTGAATGCAGTTGCTTCAGTACCCATAATGTATTCTTGGTCTCCCCTACAGCAGAATTTTATGGTGGAAGATGAAACTGTTTTACATAACATTCCTTATATGGGAGATGAAGTTTTAGATCAGGATGGTACTTTCATTGAAGAACTAATAAAAAATTATGATGGGAAAGTACACGGGGATAGAGAATGTGGGTTTATAAATGATGAAATTTTTGTGGAGTTGGTGAATGCCCTTGGTCAATATAATGATGATGACGATGATGATGATGGAGACGATCCTGAAGAAAGAGAAGAAAAGCAGAAAGATCTGGAGGATCACCGAGATGATAAAGAAAGCCGCCCACCTCGGAAATTTCCTTCTGATAAAATTTTTGAAGCCATTTCCTCAATGTTTCCAGATAAGGGCACAGCAGAAGAACTAAAGGAAAAATATAAAGAACTCACCGAACAGCAGCTCCCAGGCGCACTTCCTCCTGAATGTACCCCCAACATAGATGGACCAAATGCTAAATCTGTTCAGAGAGAGCAAAGCTTACACTCCTTTCATACGCTTTTCTGTAGGCGATGTTTTAAATATGACTGCTTCCTACATCCTTTTCATGCAACACCCAACACTTATAAGCGGAAGAACACAGAAACAGCTCTAGACAACAAACCTTGTGGACCACAGTGTTACCAGCATTTGGAGGGAGCAAAGGAGTTTGCTGCTGCTCTCACCGCTGAGCGGATAAAGACCCCACCAAAACGTCCAGGAGGCCGCAGAAGAGGACGGCTTCCCAATAACAGTAGCAGGCCCAGCACCCCCACCATTAATGTGCTGGAATCAAAGGATACAGACAGTGATAGGGAAGCAGGGACTGAAACGGGGGGAGAGAACAATGATAAAGAAGAAGAAGAGAAGAAAGATGAAACTTCGAGCTCCTCTGAAGCAAATTCTCGGTGTCAAACACCAATAAAGATGAAGCCAAATATTGAACCTCCTGAGAATGTGGAGTGGAGTGGTGCTGAAGCCTCAATGTTTAGAGTCCTCATTGGCACTTACTATGACAATTTCTGTGCCATTGCTAGGTTAATTGGGACCAAAACATGTAGACAGGTGTATGAGTTTAGAGTCAAAGAATCTAGCATCATAGCTCCAGCTCCCGCTGAGGATGTGGATACTCCTCCAAGGAAAAAGAAGAGGAAACACCGGTTGTGGGCTGCACACTGCAGAAAGATACAGCTGAAAAAGGGTCAAAACCGCTTTCCGGGATGCCGCTGCAAAGCACAGTGCAACACCAAGCAGTGCCCGTGCTACCTGGCTGTCCGAGAGTGTGACCCTGACCTCTGTCTTACTTGTGGAGCCGCTGACCATTGGGACAGTAAAAATGTGTCCTGCAAGAACTGCAGTATTCAGCGGGGCTCCAAAAAGCATCTATTGCTGGCACCATCTGACGTGGCAGGCTGGGGGATTTTTATCAAAGATCCTGTGCAGAAAAATGAATTCATCTCAGAATACTGTGGAGAGATTATTTCTCAAGATGAAGCTGACAGAAGAGGGAAAGTGTATGATAAATACATGTGCAGCTTTCTGTTCAACTTGAACAATGATTTTGTGGTGGATGCAACCCGCAAGGGTAACAAAATTCGTTTTGCAAATCATTCGGTAAATCCAAACTGCTATGCAAAAGTTATGATGGTTAACGGTGATCACAGGATAGGTATTTTTGCCAAGAGAGCCATCCAGACTGGCGAAGAGCTGTTTTTTGATTACAGATACAGCCAGGCTGATGCCCTGAAGTATGTCGGCATCGAAAGAGAAATGGAAATCCCTTGACATCTGCTACCTCCTCCCCCCTCCTCTGAAACAGCTGCCTTAGCTTCAGGAACCTCGAGTACTGTGGGCAATTTAGAAAAAGAACATGCAGTTTGAAATTCTGAATTTGCAAAGTACTGTAAGAATAATTTATAGTAATGAGTTTAAAAATCAACTTTTTATTGCCTTCTCACCAGCTGCAAAGTGTTTTGTACCAGTGAATTTTTGCAATAATGCAGTATGGTACATTTTTCAACTTTGAATAAAGAATACTTGAACTTGTCCTTGTTGAATC

A structure model of partial EZH2 protein based on the A chain ofnuclear receptor binding SET domain protein 1 (NSD1) is provided below.This model corresponds to amino acid residues 533-732 of EZH2 sequenceof SEQ ID NO: 1.

The corresponding amino acid sequence of this structure model isprovided below. The residues in the substrate pocket domain areunderlined. The residues in the SET domain are shown italic.

(SEQ ID NO: 6)SCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKK

 

The catalytic site of EZH2 is believed to reside in a conserved domainof the protein known as the SET domain. The amino acid sequence of theSET domain of EZH2 is provided by the following partial sequencespanning amino acid residues 613-726 of Swiss-Prot Accession No. Q15910(SEQ ID NO: 1):

(SEQ ID NO: 7)HLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDY.

The tyrosine (Y) residue shown underlined in SEQ ID NO: 7 is Tyr641(Y641) in Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1).

The SET domain of GenBank Accession No. NP_(—)004447 (SEQ ID NO: 3)spans amino acid residues 618-731 and is identical to SEQ ID NO:6. Thetyrosine residue corresponding to Y641 in Swiss-Prot Accession No.Q15910 shown underlined in SEQ ID NO: 7 is Tyr646 (Y646) in GenBankAccession No. NP_(—)004447 (SEQ ID NO: 3).

The SET domain of GenBank Accession No. NP_(—)694543 (SEQ ID NO: 5)spans amino acid residues 574-687 and is identical to SEQ ID NO: 7. Thetyrosine residue corresponding to Y641 in Swiss-Prot Accession No.Q15910 shown underlined in SEQ ID NO: 7 is Tyr602 (Y602) in GenBankAccession No. NP_(—)694543 (SEQ ID NO: 5).

The nucleotide sequence encoding the SET domain of GenBank Accession No.NP_(—)004447 is

(SEQ ID NO: 8)catctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattac,where the codon encoding Y641 is shown underlined.

For purposes of this application, amino acid residue Y641 of human EZH2is to be understood to refer to the tyrosine residue that is orcorresponds to Y641 in Swiss-Prot Accession No. Q15910.

Full amino acid sequence of Y641 mutant EZH2 (SEQ ID NO: 9)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEXCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPWherein x can be any amino acid residue other than tyrosine (Y)

Also for purposes of this application, a Y641 mutant of human EZH2, and,equivalently, a Y641 mutant of EZH2, is to be understood to refer to ahuman EZH2 in which the amino acid residue corresponding to Y641 ofwild-type human EZH2 is substituted by an amino acid residue other thantyrosine.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a single amino acid residue corresponding to Y641 ofwild-type human. EZH2 by an amino acid residue other than tyrosine.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of phenylalanine (F) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641F mutant or,equivalently, Y641F.

Y641F (SEQ ID NO: 10)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEFCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of histidine (H) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641H mutant or,equivalently, Y641H.

Y641H (SEQ ID NO: 11)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEHCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of asparagine (N) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641N mutant or,equivalently, Y641N.

Y641N (SEQ ID NO: 12)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISENCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of serine (S) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641S mutant or,equivalently, Y641S.

Y641S (SEQ ID NO: 13)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISESCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of cysteine (C) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641C mutant or,equivalently, Y641C.

Y641C (SEQ ID NO: 14)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISECCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP

In one embodiment the amino acid sequence of a A677 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a non-alanine amino acid, preferably glycine (G) for thesingle amino acid residue corresponding to A677 of wild-type human EZH2.The A677 mutant of EZH2 according to this embodiment is referred toherein as an A677 mutant, and preferably an A677G mutant or,equivalently, A677G.

A677 (SEQ ID NO: 15)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDXTRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP Wherein X is preferably a glycine (G).

In one embodiment the amino acid sequence of a A687 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a non-alanine amino acid, preferably valine (V) for thesingle amino acid residue corresponding to A687 of wild-type human EZH2.The A687 mutant of EZH2 according to this embodiment is referred toherein as an A687 mutant and preferably an A687V mutant or,equivalently, A687V.

A687 (SEQ ID NO: 16)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFXNHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIP Wherein X is preferably a valine (V).

In one embodiment the amino acid sequence of a R685 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a non-arginine amino acid, preferably histidine (H) orcysteine (C) for the single amino acid residue corresponding to R685 ofwild-type human EZH2. The R685 mutant of EZH2 according to thisembodiment is referred to herein as an R685 mutant and preferably anR685C mutant or an R685H mutant or, equivalently, R685H or R685C.

A685 (SEQ ID NO: 17)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIXFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPWherein X is preferably a cysteine (C) or a histidine (H).

In one embodiment the amino acid sequence of a mutant of EZH2 differsfrom the amino acid sequence of wild-type human EZH2 in one or moreamino acid residues in its substrate pocket domain as defined in SEQ IDNO: 6. The mutant of EZH2 according to this embodiment is referred toherein as an EZH2 mutant.

Mutant EZH2 comprising one or more mutations in the substratepocket domain (SEQ ID NO: 18)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRIPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEXCGEIISQDEADRRGKVYDKYMXXXLXNLNNDFXXDXTRKGNKXXXXHSVNPNCYAKVMMVNGDHRXGIFAKRAIQTGEELFXDXRYSXADALKYVGIEREMEIPWherein X can be any amino acid except the corresponding wildtype residue.

The implications of the present results for human disease are made clearby the data summarized in Table 2 (see Example 5). Cells heterozygousfor EZH2 would be expected to display a malignant phenotype due to theefficient formation of H3-K27me1 by the WT enzyme and the efficient,subsequent transition of this progenitor species to H3-K27me2, and,especially, H3-K27me3, by the mutant enzyme form(s).

It has been reported that H3-K27me1 formation is not exclusivelydependent on WT-EZH2 catalysis. Knockout studies of EZH2 and of anotherPRC2 subunit, EED, have demonstrated H3-K27me1 formation can becatalyzed by PRC2 complexes containing either EZH2 or the relatedprotein EZH1 as the catalytic subunit. Shen, X. et al. (2008) Mol Cell32:491-502. Hence, catalytic coupling between the mutant EZH2 speciesand PRC2 complexes containing either WT-EZH2 or WT-EZH1 would suffice toaugment H3-K27me2/3 formation, and thus produce the attendant malignantphenotype. The data therefore suggest that the malignant phenotype offollicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) ofthe germinal center B cell (GCB) subtype, associated with expression ofmutant forms of EZH2, is the result of an overall gain of function withrespect to formation of the trimethylated form of H3-K27. Thisinterpretation of the data also helps to reconcile the existence ofcancer-associated overexpression of EZH2 or PRC2 associated proteins(e.g., PHF19/PCL3) and also loss-of-function genotypes for the histoneH3-K27 demethylase UTX. Loss of UTX activity would be enzymaticallyequivalent to a gain of function for EZH2, in either situation resultingin greater steady state levels of tri-methylated H3-K27 in cancer cells(FIG. 4).

The mono-, di-, and tri-methylation states of histone H3-K27 areassociated with different functions in transcriptional control. HistoneH3-K27 monomethylation is associated with active transcription of genesthat are poised for transcription. Cui et al. (2009) Cell Stem Cell4:80-93; Barski (2007) Cell 129:823-37. In contrast, trimethylation ofhistone H3-K27 is associated with either transcriptionally repressedgenes or genes that are poised for transcription when histone H3-K4trimethylation is in cis. Cui et al. (supra); Kirmizis et al. (2007)Genes Dev 18:1592-1605; Bernstein et al. (2006) Cell 125:315-26. Takentogether, alterations in the PRC2 complex activity reported in cancer,including the Y641 mutation of EZH2, are predicted to result in anincrease in the trimethylated state of histone H3-K27 and thus to resultin transcriptional repression.

Another discovery of the present invention is that cells expressing amutant EZH2 comprising a mutation in the substrate pocket domain asdefined in SEQ ID NO: 6 are, in general, more sensitive to smallmolecule EZH2 inhibitors than cells expressing wild type (WT) EZH2.Specifically, cells expressing Y641 mutant EZH2 show reduced growing,dividing or proliferation, or even undergo apoptosis or necrosis afterthe treatment of EZH2 inhibitors. In contrast, cells expressing WT EZH2are not responsive to the anti-proliferative effect of the EZH2inhibitors (FIGS. 13 and 14). Particularly, cells expressing asubstitution mutation at amino acid position 677 of SEQ ID NO: 1 showgreater sensitivity to EZH2 inhibitors than cells expressing other EZH2mutants (Example 19).

EZH2 and other protein methyltransferases have been suggested to beattractive targets for drug discovery. Copeland et al. (2009) Nat RevDrug Discov 8:724-32; Copeland et al. (2010) Curr Opin Chem Biol14(4):505-10; Pollock et al. (2010) Drug Discovery Today: TherapeuticStrategies 6(1):71-9. The present data also suggest an experimentalstrategy for development of FL and GCB lymphoma-specific drugs. As thedifferences in substrate recognition between the WT anddisease-associated mutants derive from transition state interactions,small molecule inhibitors that selectively mimic the transition state ofthe mutant EZH2 over that of the WT enzyme should prove to be effectivein blocking H3-K27 methylation in mutation-bearing cells. Inhibitors ofthis type would be expected to display a large therapeutic index, astarget-mediated toxicity would be minimal for any cells bearing only theWT enzyme. Transition state mimicry has proved to be an effectivestrategy for drug design in many disease areas. See, for example,Copeland, R. A. Enzymes: A Practical Introduction to Structure,Mechanism and Data Analysis. 2nd ed, (Wiley, 2000).

The present results point to a previously unrecognized, surprisingdependency on enzymatic coupling between enzymes that perform H3-K27mono-methylation and certain mutant forms of EZH2 for pathogenesis infollicular lymphoma and diffuse large B-cell lymphoma. While notintending to be bound by any one theory, it is believed the dataconstitute the first example of a human disease that is dependent onsuch coupling of catalytic activity between normal (WT) anddisease-associated mutant (such as Y641) enzymes.

An aspect of the present invention is a method for treating oralleviating a symptom of cancer or precancerous condition in a subjectby administering to a subject expressing a mutant EZH2 comprising amutation in the substrate pocket domain as defined in SEQ ID NO: 6 atherapeutically effective amount of an EZH2 inhibitor.

Another aspect of the invention is a method for inhibiting in a subjectconversion of H3-K27 to trimethylated H3-K27. The inhibition can involveinhibiting in a subject conversion of unmethylated H3-K27 tomonomethylated H3-K27, conversion of monomethylated H3-K27 todimethylated H3-K27, conversion of dimethylated H3-K27 to trimethylatedH3-K27, or any combination thereof, including, for example, conversionof monomethylated H3-K27 to dimethylated H3-K27 and conversion ofdimethylated H3-K27 to trimethylated H3-K27. As used herein,unmethylated H3-K27 refers to histone H3 with no methyl group covalentlylinked to the amino group of lysine 27. As used herein, monomethylatedH3-K27 refers to histone H3 with a single methyl group covalently linkedto the amino group of lysine 27. Monomethylated H3-K27 is also referredto herein as H3-K27me1. As used herein, dimethylated H3-K27 refers tohistone H3 with two methyl groups covalently linked to the amino groupof lysine 27. Dimethylated H3-K27 is also referred to herein asH3-K27me2. As used herein, trimethylated H3-K27 refers to histone H3with three methyl groups covalently linked to the amino group of lysine27. Trimethylated H3-K27 is also referred to herein as H3-K27me3.

Histone H3 is a 136 amino acid long protein, the sequence of which isknown. See, for example, GenBank Accession No. CAB02546, the content ofwhich is incorporated herein by reference. As disclosed further herein,in addition to full-length histone H3, peptide fragments of histone H3comprising the lysine residue corresponding to K27 of full-lengthhistone H3 can be used as substrate for EZH2 (and likewise for mutantforms of EZH2) to assess conversion of H3-K27 ml to H3-K27m2 andconversion of H3-K27m2 to H3-K27m3. In one embodiment, such peptidefragment corresponds to amino acid residues 21-44 of histone H3. Suchpeptide fragment has the amino acid sequence LATKAARKSAPATGGVKKPHRYRP(SEQ ID NO: 19).

The method of the present invention involves administering to a subjectexpressing a mutant EZH2 a therapeutically effective amount of aninhibitor of EZH2, wherein the inhibitor inhibits histonemethyltransferase activity of EZH2, thereby inhibiting conversion ofH3-K27 to trimethylated H3-K27 in the subject and thus treating orallievating a symptom of cancer or disorders associated with abnormalhiston methylation levels in the subject.

A subject expressing a mutant EZH2 of the present invention refers to asubject having a detectable amount of a mutant EZH2 polypeptide.Preferably the mutant EZH2 polypeptide comprises one or more mutationsin its substrate pocket domain as defined in SEQ ID NO: 6. Exemplarymutation includes a substitution at amino acid position 677, 687, 674,685, or 641 of SEQ ID NO: 1, such as, but is not limited to asubstitution of glycine (G) for the wild type residue alanine (A) atamino acid position 677 of SEQ ID NO: 1 (A677G); a substitution ofvaline (V) for the wild type residue alanine (A) at amino acid position687 of SEQ ID NO: 1 (A687V); a substitution of methionine (M) for thewild type residue valine (V) at amino acid position 674 of SEQ ID NO: 1(V674M); a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685H); asubstitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 685 of SEQ ID NO: 1 (R685C); a substitution ofphenylalanine (F) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641F); a substitution of histidine (H)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641H); a substitution of asparagine (N) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641N);a substitution of serine (S) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641S); or a substitution ofcysteine (C) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641C). More preferablly, the subject hasa detectable amount of a mutant EZH2 polypeptide comprising asubstitution of glycine (G) for the wild type residue alanine (A) atamino acid position 677 of SEQ ID NO: 1 (A677G); a substitution ofvaline (V) for the wild type residue alanine (A) at amino acid position687 of SEQ ID NO: 1 (A687V); a substitution of histidine (H) for thewild type residue arginine (R) at amino acid position 685 of SEQ ID NO:1 (R685H); or a substitution of cysteine (C) for the wild type residuearginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685C).

Alternatively a subject expressing a mutant EZH2 of the presentinvention refers to a subject having a detectable amount of a nucleicacid sequence encoding a mutant EZH2 polypeptide. Preferably a nucleicacid sequence encoding a mutant EZH2 polypeptide comprises one or moremutations in its substrate pocket domain as defined in SEQ ID NO: 6.Exemplary mutation includes a substitution at amino acid position 677,687, 674, 685, or 641 of SEQ ID NO: 1, such as, but is not limited to asubstitution of glycine (G) for the wild type residue alanine (A) atamino acid position 677 of SEQ ID NO: 1 (A677G); a substitution ofvaline (V) for the wild type residue alanine (A) at amino acid position687 of SEQ ID NO: 1 (A687V); a substitution of methionine (M) for thewild type residue valine (V) at amino acid position 674 of SEQ ID NO: 1(V674M); a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685H); asubstitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 685 of SEQ ID NO: 1 (R685C); a substitution ofphenylalanine (F) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641F); a substitution of histidine (H)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641H); a substitution of asparagine (N) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641N);a substitution of serine (S) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641S); or a substitution ofcysteine (C) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641C). More preferably, the subject has adetectable amount of nucleic acid sequence encoding a mutant EZH2polypeptide comprising a substitution of glycine (G) for the wild typeresidue alanine (A) at amino acid position 677 of SEQ ID NO: 1 (A677G);a substitution of valine (V) for the wild type residue alanine (A) atamino acid position 687 of SEQ ID NO: 1 (A687V); a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 685 of SEQ ID NO: 1 (R685H); or a substitution of cysteine (C)for the wild type residue arginine (R) at amino acid position 685 of SEQID NO: 1 (R685C).

Detection of EZH2 Mutants

A mutant EZH2 polypeptide can be detected using any suitable method. Forexample, a mutant EZH2 polypeptide can be detected using an antibodythat binds specifically to the mutant EZH2 polypeptide or to a peptidefragment that is characteristic of the mutant EZH2 polypeptide. Apeptide fragment that is characteristic of the mutant EZH2 polypeptidemay include, for example, a SET domain as provided in SEQ ID NO: 7,except for substitution of one or more residues in the substrate pocketdomain as defined in SEQ ID NO: 6 by an amino acid residue other thanthe wild type residue. In another embodiment, a peptide fragment that ischaracteristic of the mutant EZH2 polypeptide may include, for example,a 10-113 amino acid fragment of the SET domain as provided in SEQ ID NO:7, except for substitution of one or more residues in the substratepocket domain as defined in SEQ ID NO: 6 by an amino acid residue otherthan the wild type residue, provided that the fragment includes theamino acid residue corresponding to a mutation of EZH2. An antibody isconsidered to bind specifically to the mutant EZH2 polypeptide or to apeptide fragment that is characteristic of the mutant EZH2 polypeptideif it binds to that mutant EZH2 polypeptide or peptide fragment thereofbut not to the corresponding wild-type EZH2 polypeptide or peptidefragment thereof. In one embodiment, such antibody is considered to bindspecifically to the mutant EZH2 polypeptide or to a peptide fragmentthat is characteristic of the mutant EZH2 polypeptide if it binds tothat mutant EZH2 polypeptide or peptide fragment thereof with anaffinity that is at least ca. 100-fold greater than for thecorresponding wild-type EZH2 polypeptide or peptide fragment thereof. Inone embodiment, such antibody is considered to bind specifically to themutant EZH2 polypeptide or to a peptide fragment that is characteristicof the mutant EZH2 polypeptide if it binds to that mutant EZH2polypeptide or peptide fragment thereof with an affinity that is atleast ca. 1000-fold greater than for the corresponding wild-type EZH2polypeptide or peptide fragment thereof. The antibody can be used, forexample, in an enzyme-linked immunosorbent assay (ELISA) or Western blotassay. The antibody may be monoclonal, polyclonal, chimeric, or anantibody fragment. The step of detecting the reaction product may becarried out with any suitable immunoassay.

In one embodiment the antibody is a monoclonal antibody. A monoclonalantibody can be prepared according to conventional methods well known inthe art. See, for example, Köhler and Milstein (1975) Nature 256(5517):495-7.

As another example, a mutant EZH2 polypeptide can be detected using massspectrometry (MS), e.g., electrospray ionization coupled withtime-of-flight (ESI-TOF) or matrix-assisted laser desorption/ionizationcoupled with time-of-flight (MALDI-TOF). Such methods are well known inthe art. The analysis will involve identification of one or more peptidefragments comprising the mutation of interest, for example, a peptide 12to 24 amino acids long comprising a sequence spanning the amino acidcorresponding to a mutation in wild-type EZH2.

A nucleic acid sequence encoding a mutant EZH2 polypeptide or a peptidefragment that is characteristic of the mutant EZH2 polypeptide can bedetected using any suitable method. For example, a nucleic acid sequenceencoding a mutant EZH2 polypeptide can be detected using whole-genomeresequencing or target region resequencing (the latter also known astargeted resequencing) using suitably selected sources of DNA andpolymerase chain reaction (PCR) primers in accordance with methods wellknown in the art. See, for example, Bentley (2006) Curr Opin Genet Dev.16:545-52, and Li et al. (2009) Genome Res 19:1124-32. The methodtypically and generally entails the steps of genomic DNA purification,PCR amplification to amplify the region of interest, cycle sequencing,sequencing reaction cleanup, capillary electrophoresis, and dataanalysis. High quality PCR primers to cover region of interest aredesigned using in silico primer design tools. Cycle sequencing is asimple method in which successive rounds of denaturation, annealing, andextension in a thermal cycler result in linear amplification ofextension products. The products are typically terminated with afluorescent tag that identifies the terminal nucleotide base as G, A, T,or C. Unincorporated dye terminators and salts that may compete forcapillary eletrophoretic injection are removed by washing. Duringcapillary electrophoresis, the products of the cycle sequencing reactionmigrate through capillaries filled with polymer. The negatively chargedDNA fragments are separated by size as they move through the'capillariestoward the positive electrode. After electrophoresis, data collectionsoftware creates a sample file of the raw data. Using downstreamsoftware applications, further data analysis is performed to translatethe collected color data images into the corresponding nucleotide bases.Alternatively or in addition, the method may include the use ofmicroarray-based targeted region genomic DNA capture and/or sequencing.Kits, reagents, and methods for selecting appropriate PCR primers andperforming resequencing are commercially available, for example, fromApplied Biosystems, Agilent, and NimbleGen (Roche Diagnostics GmbH).Methods such as these have been used to detect JAK2 andmyeloproliferative leukemia gene (MPL) mutations and to diagnosepolycythemia vera, essential thrombocythemia, and idiopathicmyelofibrosis. For use in the instant invention, PCR primers may beselected so as to amplify, for example, at least a relevant portion ofSEQ ID NO: 8 (above).

Alternatively or in addition, a nucleic acid sequence encoding a mutantEZH2 polypeptide may be detected using a Southern blot in accordancewith methods well known in the art. In one embodiment a DNA sequenceencoding a mutant EZH2 polypeptide is detected using nucleic acidhybridization performed under highly stringent conditions. A nucleicacid probe is selected such that its sequence is complementary to atarget nucleic acid sequence that includes a codon for the mutant aminoacid corresponding to a mutation of wild-type EZH2. Such mutationincludes any mutation of EZH2, preferably one or more mutations in thesubstrate pocket domain as defined in SEQ ID NO: 6 of EZH2, such as, butare not limited to a substitution of glycine (G) for the wild typeresidue alanine (A) at amino acid position 677 of SEQ ID NO: 1 (A677G);a substitution of valine (V) for the wild type residue alanine (A) atamino acid position 687 of SEQ ID NO: 1 (A687V); a substitution ofmethionine (M) for the wild type residue valine (V) at amino acidposition 674 of SEQ ID NO: 1 (V674M); a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 685 of SEQID NO: 1 (R685H); a substitution of cysteine (C) for the wild typeresidue arginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685C);a substitution of phenylalanine (F) for the wild type residue tyrosine(Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F); a substitutionof histidine (H) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641H); a substitution of asparagine (N)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641N); a substitution of serine (S) for the wild type residuetyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641S); or asubstitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641C).

A sequence-specific probe is combined with a sample to be tested underhighly stringent conditions. The term “highly stringent conditions” asused herein refers to parameters with which the art is familiar. Nucleicacid hybridization parameters may be found in references that compilesuch methods, e.g., J. Sambrook, et al., eds., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989, or F. M. Ausubel, et al., eds., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., New York. Morespecifically, highly stringent conditions, as used herein, refers, forexample, to hybridization at 65° C. in hybridization buffer (3.5×SSC,0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin(BSA), 2.5 mM NaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodiumchloride/0.015 M sodium citrate, pH 7; SDS is sodium dodecyl sulphate;and EDTA is ethylenediaminetetracetic acid. After hybridization, themembrane upon which the DNA is transferred is washed, for example, in2×SSC at room temperature and then at 0.1-0.5×SSC/0.1×SDS attemperatures up to 68° C.

There are other conditions, reagents, and so forth that can be used,which result in a similar degree of stringency. The skilled artisan willbe familiar with such conditions, and thus they are not given here. Itwill be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof EZH2-associated nucleic acids of the invention, including, inparticular, nucleic acids encoding mutants of EZH2 (e.g., by using lowerstringency conditions). The skilled artisan also is familiar with themethodology for screening cells and libraries for expression of suchmolecules, which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing.

The subject is administered a therapeutically effective amount of aninhibitor of EZH2. As used herein, an inhibitor of EZH2 refers,generally, to a small molecule, i.e., a molecule of molecular weightless than about 1.5 kilodaltons (kDa), which is capable of interferingwith the histone methyltransferase enzymatic activity of EZH2.

In one embodiment the inhibitor of EZH2 inhibits histonemethyltransferase activity of wild-type EZH2. In one embodiment theinhibitor of EZH2 inhibits histone methyltransferase activity of themutant EZH2. In one embodiment the inhibitor of EZH2 inhibits histonemethyltransferase activity of wild-type EZH2 and histonemethyltransferase activity of the mutant EZH2. In one embodiment theinhibitor of EZH2 selectively inhibits histone methyltransferaseactivity of the mutant EZH2.

As disclosed herein, certain mutants of EZH2 (such as Y641) arerelatively poor catalysts for conversion of unmethylated H3-K27 toH3-K27me1 and yet unexpectedly effective catalysts for conversion ofH3-K27me2 to H3-K27me3. Certain mutants of EZH2 (such as A687) prefermonomethyl H3-K27 substrate. In contrast, certain mutants of EZH2 (suchas A677) show equal preference among unmethylated, monomethylated anddimethylated H3-K27. Conversely, wild-type EZH2 is a relativelyeffective catalyst for conversion of unmethylated H3-K27 to H3-K27me1and yet unexpectedly ineffective catalyst for conversion of H3-K27me2 toH3-K27me3. This is important because mono-, di- and tri-methylatedstates of H3-K27 exhibit different functions in transcriptional control.For example, H3-K27me1 is associated with active transcription of genesthat are poised for transcription, while H3-K27me3 is associated withtranscriptionally repressed genes or genes that are poised fortranscription when H3-K4 trimethylation is in cis. Thus, selectiveinhibition of histone methyltransferase activity of the mutant of EZH2affects selective inhibition of production of the different methylatedforms of H3-K27, thereby modifying transcription associated with H3-K27methylation levels.

An inhibitor of EZH2 “selectively inhibits” histone methyltransferaseactivity of the mutant EZH2 when it inhibits histone methyltransferaseactivity of the mutant EZH2 more effectively than it inhibits histonemethyltransferase activity of wild-type EZH2. For example, in oneembodiment the selective inhibitor has an IC50 for the mutant EZH2 thatis at least 40 percent lower than the IC50 for wild-type EZH2. In oneembodiment the selective inhibitor has an IC50 for the mutant EZH2 thatis at least 50 percent lower than the IC50 for wild-type EZH2. In oneembodiment the selective inhibitor has an IC50 for the mutant EZH2 thatis at least 60 percent lower than the IC50 for wild-type EZH2. In oneembodiment the selective inhibitor has an IC50 for the mutant EZH2 thatis at least 70 percent lower than the IC50 for wild-type EZH2. In oneembodiment the selective inhibitor has an IC50 for the mutant EZH2 thatis at least 80 percent lower than the IC50 for wild-type EZH2. In oneembodiment the selective inhibitor has an IC50 for the mutant EZH2 thatis at least 90 percent lower than the IC50 for wild-type EZH2.

In one embodiment, the selective inhibitor of a mutant EZH2 exertsessentially no inhibitory effect on wild-type EZH2.

The inhibitor inhibits conversion of H3-K27me2 to H3-K27me3. In oneembodiment the inhibitor is said to inhibit trimethylation of H3-K27.Since conversion of H3-K27me1 to H3-K27me2 precedes conversion ofH3-K27me2 to H3-K27me3, an inhibitor of conversion of H3-K27me1 toH3-K27me2 naturally also inhibits conversion of H3-K27me2 to H3-K27me3,i.e., it inhibits trimethylation of H3-K27. It is also possible toinhibit conversion of H3-K27me2 to H3-K27me3 without inhibition ofconversion of H3-K27me1 to H3-K27me2. Inhibition of this type would alsoresult in inhibition of trimethylation of H3-K27, albeit withoutinhibition of dimethylation of H3-K27.

In one embodiment the inhibitor inhibits conversion of H3-K27me1 toH3-K27me2 and the conversion of H3-K27me2 to H3-K27me3. Such inhibitormay directly inhibit the conversion of H3-K27me1 to H3-K27me2 alone.Alternatively, such inhibitor may directly inhibit both the conversionof H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3.

The inhibitor inhibits histone methylase activity. Inhibition of histonemethylase activity can be detected using any suitable method. Theinhibition can be measured, for example, either in terms of rate ofhistone methylase activity or as product of histone methylase activity.Methods suitable for either of these readouts are included in theExamples below.

The inhibition is a measurable inhibition compared to a suitablenegative control. In one embodiment, inhibition is at least 10 percentinhibition compared to a suitable negative control. That is, the rate ofenzymatic activity or the amount of product with the inhibitor is lessthan or equal to 90 percent of the corresponding rate or amount madewithout the inhibitor. In various other embodiments, inhibition is atleast 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 95 percent inhibitioncompared to a suitable negative control. In one embodiment, inhibitionis at least 99 percent inhibition compared to a suitable negativecontrol. That is, the rate of enzymatic activity or the amount ofproduct with the inhibitor is less than or equal to 1 percent of thecorresponding rate or amount made without the inhibitor.

In one embodiment, the inhibitor is S-adenosyl-L-homocysteine (SAH). SAHhas the structural formula

and is commercially available from a number of suppliers, including, forexample, Sigma-Aldrich, St. Louis, Mo. SAH has been described as aninhibitor of transmethylation by S-adenosylmethionine-dependentmethyltransferases.

In one embodiment, the inhibitor is Compound 75

or a pharmaceutically acceptable salt thereof.

In certain embodiments the invention comprises the step of performing anassay to detect a mutant of EZH2 in a sample from a subject. Assays ofthis type are described above. As used herein, a “sample from a subject”refers to any suitable sample containing cells or components of cellsobtained or derived from a subject. In one embodiment the sampleincludes cells suspected to express mutant EZH2, e.g., cancer cells. Inone embodiment the sample is a blood sample. In one embodiment thesample is a biopsy sample obtained from, for example, a lymphatic tissue(e.g., lymph node) or bone marrow. In one embodiment the sample is abiopsy sample obtained from a tissue other than or in addition to alymphatic tissue (e.g., lymph node) or bone marrow. For example, in oneembodiment the sample is a biopsy from a cancer, e.g., a tumor composedof cancer cells. Cells in the sample can be isolated from othercomponents of the sample. For example, peripheral blood mononuclearcells (PBMCs) can be isolated as a buffy coat from a blood sample thathas been centrifuged in accordance with methods familiar to those ofskill in the art.

When the result of the assay on a sample from a subject indicates that amutant EZH2 is present in the sample, the subject is said to expressmutant EZH2. Indeed, in one embodiment, when the result of the assay ona sample from a subject indicates that a mutant EZH2 is present in thesample, the subject is identified as a candidate for treatment with aninhibitor of EZH2, wherein the inhibitor selectively inhibits histonemethyltransferase activity of the mutant EZH2.

When the result of the assay on a sample from a cancer indicates that amutant EZH2 is present in the cancer, the cancer is said to express themutant EZH2.

Similarly, when the result of the assay on a sample comprising cancercells from a subject having a cancer indicates that a mutant EZH2 ispresent in the sample, the subject is said to express the mutant EZH2.

Detection of dimethylated H3-K27 or trimethlated H3-K27 can beaccomplished using any suitable method in the art. In one embodiment,the methylation level is detected using antibodies specific fordimethylated H3-K27 or trimethlated H3-K27. For example, the isolatedtissue is formalin fixed and embedded in paraffin blocks for long termpreservation. The blocks can be used to prepare slides forimmunohistochemical staining or fluorescent staining with antibodiesagainst methylated H3-K27. Alternatively, whole cell lysates or histoneextracts can be prepared from the isolated tissue sample andsubsequently used for immunohistochemical staining, western blotanalysis or fluorescent staining. In another embodiment the methylationlevel is detected using a polypeptide or an aptamer specific fordimethylated H3-K27 or trimethlated H3-K27. In another embodiment, themethylation level is detected using mass spectrometry (MS).

A control dimethylated H3-K27 or a control trimethlated H3-K27 can beestablished from a control sample, e.g., an adjacent non-tumor tissueisolated from the subject or a healthy tissue from a healthy subject.Alternatively, the control methylation level of H3-K27me2 or H3-K27me3can be established by a pathologist with known methods in the art.

Screening Methods

An aspect of the invention is a method for identifying a test compoundas an inhibitor of a mutant EZH2. In one embodiment the method includescombining an isolated mutant EZH2 with a histone substrate, a methylgroup donor (such as S-adenosyl methionine (SAM)), and a test compound,wherein the histone substrate comprises a form of H3-K27 selected fromthe group consisting of unmethylated H3-K27, monomethylated H3-K27,dimethylated H3-K27, and any combination thereof; and performing anassay to detect methylation of H3-K27 in the histone substrate, therebyidentifying the test compound as an inhibitor of the mutant EZH2 whenmethylation of H3-K27 in the presence of the test compound is less thanmethylation of H3-K27 in the absence of the test compound. The assay todetect methylation of H3-K27 can be selected to measure the rate ofmethylation, the extent of methylation, or both the rate and extent ofmethylation.

The mutant EZH2 is isolated as a PRC2 complex or functional equivalentthereof. As used herein, the term “isolated” means substantiallyseparated from other components with which the complex may be found asit occurs in nature. A compound can be isolated without necessarilybeing purified. In one embodiment the mutant of EZH2 is isolated as acomplex of a mutant EZH2 together with EED and SUZ12. In anotherembodiment the mutant of EZH2 is isolated as a complex of a mutant EZH2together with EED, SUZ12, and RbAp48. Under appropriate conditions, aPRC2 complex or functional equivalent thereof exhibits histonemethyltransferase activity for H3-K27. In one embodiment the complex iscomposed of recombinantly expressed component polypeptides, e.g., EZH2,EED, SUZ12, with or without RbAp48.

The isolated mutant EZH2 is combined with a histone substrate. A histonesubstrate includes any suitable source of histone polypeptides orfragments thereof that can serve as substrate for EZH2. In oneembodiment the histone substrate includes histones isolated from asubject. The histones can be isolated from cells of a subject using anysuitable method; such methods are well known to persons skilled in theart and need not be further specified here. See, for example, Fang etal. (2004) Methods Enzymol 377:213-26. In accordance with the Examplesbelow, in one embodiment the histone substrate is provided asnucleosomes. In accordance with the Examples below, in one embodimentthe histone substrate is provided as avian (chicken) erythrocytenucleosomes.

Histone substrate so provided may include an admixture of states ofhistone modification, including various states of H3-K27 methylation asjudged by Western blotting with H3-K27 methylation state-specificantibodies. In one embodiment the histone substrate may be provided aspurified full-length histone H3. Such purified full-length histone H3may be provided as a homogeneous preparation in respect of states ofH3-K27 methylation, or as an admixture of various states of H3-K27methylation. Homogeneous preparations of isolated histone H3 in respectof states of H3-K27 methylation may be prepared in part by passage overan immunoaffinity column loaded with suitable H3-K27 methylationstate-specific antibodies or by immunoprecipitation using magnetic beadscoated with suitable H3-K27 methylation state-specific antibodies.Alternatively or in addition, the methylation state of H3-K27 can becharacterized as part of performing the assay. For example, the startingmaterial histone substrate might be characterized as containing 50percent unmethylated H3-K27, 40 percent monomethylated H3-K27, 10percent dimethylated H3-K27, and 0 percent trimethylated H3-K27.

In one embodiment the histone substrate includes a peptide library or asuitable peptide comprising one or more amino acid sequences related tohistone H3, including, in particular, a sequence that encompassesH3-K27. For example, in one embodiment, the histone substrate is apeptide fragment that corresponds to amino acid residues 21-44 ofhistone H3. Such peptide fragment has the amino acid sequenceLATKAARKSAPATGGVKKPHRYRP (SEQ ID NO: 19). The peptide library or peptidecan be prepared by peptide synthesis according to techniques well knownin the art and optionally modified so as to incorporate any desireddegree of methylation of lysine corresponding to H3-K27. As described inthe Examples below, such peptides can also be modified to incorporate alabel, such as biotin, useful in performing downstream assays. In oneembodiment the label is appended to the amino (N)-terminus of thepeptide(s). In one embodiment the label is appended to the carboxy(C)-terminus of the peptide(s).

H3-K27 methylation-specific antibodies are available from a variety ofcommercial sources, including, for example, Cell Signaling Technology(Danvers, Mass.) and Active Motif (Carlsbad, Calif.).

The isolated mutant EZH2 is combined with a test compound. As usedherein, a “test compound” refers to a small organic molecule having amolecular weight of less than about 1.5 lcDa. In one embodiment a testcompound is a known compound. In one embodiment a test compound is anovel compound. In one embodiment, a test compound can be provided aspart of a library of such compounds, wherein the library includes, forexample, tens, hundreds, thousands, or even more compounds. A library ofcompounds may advantageously be screened in a high throughput screeningassay, for example, using arrays of test compounds and roboticmanipulation in accordance with general techniques well known in theart.

In certain embodiments a test compound is a compound that is aderivative of SAH or a derivative of Compound 75.

Detection of methylation of H3-K27 can be accomplished using anysuitable method. In one embodiment, the source of donor methyl groupsincludes methyl groups that are labeled with a detectable label. Thedetectable label in one embodiment is an isotopic label, e.g., tritium.Other types of labels may include, for example, fluorescent labels.

Detection of formation of trimethylated H3-K27 can be accomplished usingany suitable method. For example, detection of formation oftrimethylated H3-K27 can be accomplished using an assay to detectincorporation of labeled methyl groups, such as described above,optionally combined with a chromatographic or other method to separatelabeled products by size, e.g., polyacrylamide gel electrophoresis(PAGE), capillary electrophoresis (CE), or high pressure liquidchromatography (HPLC). Alternatively or in addition, detection offormation of trimethylated H3-K27 can be accomplished using antibodiesthat are specific for trimethylated H3-K27.

Detection of conversion of monomethylated H3-K27 to dimethylated H3-K27can be accomplished using any suitable method. In one embodiment theconversion is measured using antibodies specific for monomethylatedH3-K27 and dimethylated H3-K27. For example, starting amounts orconcentrations of monomethylated H3-K27 and dimethylated H3-K27 may bedetermined using appropriate antibodies specific for monomethylatedH3-K27 and dimethylated H3-K27. Following the combination of enzyme,substrate, methyl group donor, and test compound, resulting amounts orconcentrations of monomethylated H3-K27 and dimethylated H3-K27 may thenbe determined using appropriate antibodies specific for monomethylatedH3-K27 and dimethylated H3-K27. The beginning and resulting amounts orconcentrations of monomethylated H3-K27 and dimethylated H3-K27 can thenbe compared. Alternatively or in addition, beginning and resultingamounts or concentrations of monomethylated H3-K27 and dimethylatedH3-K27 can then be compared to corresponding amounts of concentrationsfrom a negative control. A negative control reaction, in which no testagent is included in the assay, can be run in parallel or as ahistorical control. Results of such control reaction can optionally besubtracted from corresponding results of the experimental reaction priorto or in conjunction with making the comparison mentioned above.

Because the dimethylated form of H3-K27 may be further methylated in thesame assay, a reduction in the amount or concentration of monomethylatedH3-K27 may not appear to correspond directly to an increase indimethylated H3-K27. In this instance, it may be presumed, however, thata reduction in the amount or concentration of monomethylated H3-K27 is,by itself, reflective of conversion of monomethylated H3-K27 todimethylated H3-K27.

Detection of conversion of dimethylated H3-K27 to trimethylated H3-K27can be accomplished using any suitable method. In one embodiment theconversion is measured using antibodies specific for dimethylated H3-K27and trimethylated H3-K27. For example, starting amounts orconcentrations of dimethylated H3-K27 and trimethylated H3-K27 may bedetermined using appropriate antibodies specific for dimethylated H3-K27and trimethylated H3-K27. Following the combination of enzyme,substrate, and test compound, resulting amounts or concentrations ofdimethylated H3-K27 and trimethylated H3-K27 may then be determinedusing appropriate antibodies specific for dimethylated H3-K27 andtrimethylated H3-K27. The beginning and resulting amounts orconcentrations of dimethylated H3-K27 and trimethylated H3-K27 can thenbe compared. Alternatively or in addition, beginning and resultingamounts or concentrations of dimethylated H3-K27 and trimethylatedH3-K27 can then be compared to corresponding amounts of concentrationsfrom a negative control. A negative control reaction, in which no testagent is included in the assay, can be run in parallel or as ahistorical control. Results of such control reaction can optionally besubtracted from corresponding results of the experimental reaction priorto or in conjunction with making the comparison mentioned above.

A test agent is identified as an inhibitor of the mutant EZH2 whenmethylation of H3-K27 with the test compound is less than methylation ofH3-K27 without the test compound. In one embodiment, a test agent isidentified as an inhibitor of the mutant EZH2 when formation oftrimethylated H3-K27 in the presence of the test compound is less thanformation of trimethylated H3-K27 in the absence of the test compound.

An aspect of the invention is a method for identifying a selectiveinhibitor of a mutant EZH2. In one embodiment the method includescombining an isolated mutant EZH2 with a histone substrate, a methylgroup donor (e.g., SAM), and a test compound, wherein the histonesubstrate comprises a form of H3-K27 selected from the group consistingof monomethylated H3-K27, dimethylated H3-K27, and a combination ofmonomethylated H3-K27 and dimethylated H3-K27, thereby forming a testmixture; combining an isolated wild-type EZH2 with a histone substrate,a methyl group donor (e.g., SAM), and a test compound, wherein thehistone substrate comprises a form of H3-K27 selected from the groupconsisting of monomethylated H3-K27, dimethylated H3-K27, and acombination of monomethylated H3-K27 and dimethylated H3-K27, therebyforming a control mixture; performing an assay to detect trimethylationof the histone substrate in each of the test mixture and the controlmixture; calculating the ratio of (a) trimethylation with the mutantEZH2 and the test compound (M+) to (b) trimethylation with the mutantEZH2 without the test compound (M−); calculating the ratio of (c)trimethylation with wild-type EZH2 and the test compound (WT+) to (d)trimethylation with wild-type EZH2 without the test compound (WT−);comparing the ratio (a)/(b) with the ratio (c)/(d); and identifying thetest compound as a selective inhibitor of the mutant EZH2 when the ratio(a)/(b) is less than the ratio (c)/(d). In one embodiment the methodfurther includes taking into account a negative control without testcompound for either or both of the test mixture and the control mixture.

The present invention also provides a previously unrecognized,surprising correlation of a patient's responsiveness to an EZH2inhibitor. Accordingly, an aspect of the invention is a method fordetermining a responsiveness to an EZH2 inhibitor in a subject having acancer or a precancerous condition. The method generally involves indetecting a mutation in the EZH2 substrate pocket domain as defined inSEQ ID NO: 6. For example, the mutation may be a substitution, a pointmutation, a nonsense mutation, a miss sense mutation, a deletion, or aninsertion described above. Preferred substitution amino acid mutationincludes a substitution at amino acid position 677, 687, 674, 685, or641 of SEQ ID NO: 1, such as, but is not limited to a substitution ofglycine (G) for the wild type residue alanine (A) at amino acid position677 of SEQ ID NO: 1 (A677G); a substitution of valine (V) for the wildtype residue alanine (A) at amino acid position 687 of SEQ ID NO: 1(A687V); a substitution of methionine (M) for the wild type residuevaline (V) at amino acid position 674 of SEQ ID NO: 1 (V674M); asubstitution of histidine (H) for the wild type residue arginine (R) atamino acid position 685 of SEQ ID NO: 1 (R685H); a substitution ofcysteine (C) for the wild type residue arginine (R) at amino acidposition 685 of SEQ ID NO: 1 (R685C); a substitution of phenylalanine(F) for the wild type residue tyrosine (Y) at amino acid position 641 ofSEQ ID NO: 1 (Y641F); a substitution of histidine (H) for the wild typeresidue tyrosine (Y) at, amino acid position 641 of SEQ ID NO: 1(Y641H); a substitution of asparagine (N) for the wild type residuetyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641N); asubstitution of serine (S) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641S); or a substitution ofcysteine (C) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641C).

In a preferred embodiment, the subject has cancer or a cancerouscondition. For example, the cancer is lymphoma, leukemia or melanoma.Preferably, the lymphoma is non-Hodgkin lymphoma, follicular lymphoma ordiffuse large B-cell lymphoma. Alternatively, the leukemia is chronicmyelogenous leukemia (CML). The precancerous condition ismyelodysplastic syndromes (MDS, formerly known as preleukemia).

In another preferred embodiment, the mutant EZH2 polypeptide or thenucleic acid sequence encoding the mutant EZH2 polypeptide of thepresent invention comprises one or more mutations in the substratepocket domain of EZH2 as defined in SEQ ID NO: 6. More preferably, themutant EZH2 polypeptide or the nucleic acid sequence encoding the mutantEZH2 polypeptide of the present invention comprises a substitution atamino acid position 677, 687, 674, 685, or 641 of SEQ ID NO: 1, such as,but is not limited to a substitution of glycine (G) for the wild typeresidue alanine (A) at amino acid position 677 of SEQ ID NO: 1 (A677G);a substitution of valine (V) for the wild type residue alanine (A) atamino acid position 687 of SEQ ID NO: 1 (A687V); a substitution ofmethionine (M) for the wild type residue valine (V) at amino acidposition 674 of SEQ ID NO: 1 (V674M); a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 685 of SEQID NO: 1 (R685H); a substitution of cysteine (C) for the wild typeresidue arginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685C);a substitution of phenylalanine (F) for the wild type residue tyrosine(Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F); a substitutionof histidine (H) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641H); a substitution of asparagine (N)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641N); a substitution of serine (S) for the wild type residuetyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641S); or asubstitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641C).

Pharmaceutical Compositions

One or more EZH2 antagonists can be administered alone to a humanpatient or in pharmaceutical compositions where they are mixed withsuitable carriers or excipient(s) at doses to treat or ameliorate adisease or condition as described herein. Mixtures of these EZH2antagonists can also be administered to the patient as a simple mixtureor in suitable formulated pharmaceutical compositions. For example, oneaspect of the invention relates to pharmaceutical composition comprisinga therapeutically effective dose of an EZH2 antagonist, or apharmaceutically acceptable salt, hydrate, enantiomer or stereoisomerthereof; and a pharmaceutically acceptable diluent or carrier.

Techniques for formulation and administration of EZH2 antagonists may befound in references well known to one of ordinary skill in the art, suchas Remington's “The Science and Practice of Pharmacy,” 21st ed.,Lippincott Williams & Wilkins 2005.

Suitable routes of administration may, for example, include oral,rectal, or intestinal administration; parenteral delivery, includingintravenous, intramuscular, intraperitoneal, subcutaneous, orintramedullary injections, as well as intrathecal, directintraventricular, or intraocular injections; topical delivery, includingeyedrop and transdermal; and intranasal and other transmucosal delivery.

Alternatively, one may administer an EZH2 antagonist in a local ratherthan a systemic manner, for example, via injection of the EZH2antagonist directly into an edematous site, often in a depot orsustained release formulation.

In one embodiment, an EZH2 antagonist is administered by directinjection into a tumor or lymph node.

Furthermore, one may administer an EZH2 antagonist in a targeted drugdelivery system, for example, in a liposome coated with cancercell-specific antibody.

The pharmaceutical compositions of the present invention may bemanufactured, e.g., by conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active EZH2 antagonistsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants are used in the formulationappropriate to the barrier to be permeated. Such penetrants aregenerally known in the art.

For oral administration, the EZH2 antagonists can be formulated readilyby combining the active EZH2 antagonists with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable the EZH2antagonists of the invention to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by combining the active EZH2antagonist with a solid excipient, optionally grinding a resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active EZH2 antagonist doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active EZH2 antagonists may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols. In addition, stabilizers may be added.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the EZH2 antagonists for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the EZH2antagonist and a suitable powder base such as lactose or starch.

The EZH2 antagonists can be formulated for parenteral administration byinjection, e.g., bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active EZH2 antagonists in water-soluble form.Additionally, suspensions of the active EZH2 antagonists may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of the EZH2antagonists to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forreconstitution before use with a suitable vehicle, e.g., sterilepyrogen-free water.

The EZH2 antagonists may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases, such as cocoa butter or other glycerides.

In addition to the formulations described previously, the EZH2antagonists may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for example,subcutaneously or intramuscularly or by intramuscular injection). Thus,for example, the EZH2 antagonists may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (for example, as a sparingly soluble salt).

Alternatively, other delivery systems for hydrophobic pharmaceuticalEZH2 antagonists may be employed. Liposomes and emulsions are examplesof delivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents such as dimethysulfoxide also may be employed. Additionally,the EZH2 antagonists may be delivered using a sustained-release system,such as semi-permeable matrices of solid hydrophobic polymers containingthe therapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the EZH2 antagonists for a few weeks up to over 100 days.Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical compositions may also comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymers,such as polyethylene glycols.

Methods of Treatment

Provided herein are methods of treating, preventing or alleviating asymptom of conditions and diseases, such as cancers and precancerousconditions, the course of which can be influenced by modulating themethylation status of histones or other proteins, wherein saidmethylation status is mediated at least in part by the activity of EZH2.Modulation of the methylation status of histones can in turn influencethe level of expression of target genes activated by methylation, and/ortarget genes suppressed by methylation.

For example, one aspect of the invention relates to a method fortreating or alleviating a symptom of cancer or precancerous condition.The method comprises the step of administering to a subject having acancer or a precancerous condition and expressing a mutant EZH2 atherapeutically effective amount of an inhibitor of EZH2. Preferably, amutant EZH2 polypeptide or a nucleic acid sequence encoding a mutantEZH2 polypeptide comprises a mutation in its substrate pocket domain asdefined in SEQ ID NO: 6. More preferablly, a mutant EZH2 polypeptide ora nucleic acid sequence encoding a mutant EZH2 polypeptide comprises asubstitution mutation at amino acid position 677, 687, 674, 685, or 641of SEQ ID NO: 1, such as, but is not limited to a substitution ofglycine (G) for the wild type residue alanine (A) at amino acid position677 of SEQ ID NO: 1 (A677G); a substitution of valine (V) for the wildtype residue alanine (A) at amino acid position 687 of SEQ ID NO: 1(A687V); a substitution of methionine (M) for the wild type residuevaline (V) at amino acid position 674 of SEQ ID NO: 1 (V674M); asubstitution of histidine (H) for the wild type residue arginine (R) atamino acid position 685 of SEQ ID NO: 1 (R685H); a substitution ofcysteine (C) for the wild type residue arginine (R) at amino acidposition 685 of SEQ ID NO: 1 (R685C); a substitution of phenylalanine(F) for the wild type residue tyrosine (Y) at amino acid position 641 ofSEQ ID NO: 1 (Y641F); a substitution of histidine (H) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641H);a substitution of asparagine (N) for the wild type residue tyrosine (Y)at amino acid position 641 of SEQ ID NO: 1 (Y641N); a substitution ofserine (S) for the wild type residue tyrosine (Y) at amino acid position641 of SEQ ID NO: 1 (Y641S); or a substitution of cysteine (C) for thewild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO:1 (Y641C).

In one embodiment the inhibitor inhibits histone methyltransferaseactivity of the mutant EZH2. In one embodiment the inhibitor selectivelyinhibits histone methyltransferase activity of the mutant EZH2.Optionally, the cancer is a lymphoma. Preferably, the cancer is anon-Hodgkin lymphoma, a follicular lymphoma or a diffuse large B-celllymphoma (DLBCL). Alternatively, the cancer is leukemia (such as CML) ormelanoma. The precancerous condition includes, but is not limited to,myelodysplastic syndromes (MDS, formerly known as preleukemia).

Diseases such as cancers and neurological disease can be treated byadministration of modulators of protein (e.g., histone) methylation,e.g., modulators of histone methyltransferase, or histone demethylaseenzyme activity. Histone methylation has been reported to be involved inaberrant expression of certain genes in cancers, and in silencing ofneuronal genes in non-neuronal cells. Modulators described herein can beused to treat such diseases, i.e., to inhibit methylation of histones inaffected cells.

Based at least on the fact that abnormal histone methylation has beenfound to be associated with certain cancers and precancerous conditions,a method for treating cancer or a precancerous condition with a mutantEZH2 in a subject comprises administering to the subject in need thereofa therapeutically effective amount of a compound that inhibitsmethylation or restores methylation to roughly its level in counterpartnormal cells. In one embodiment a method for treating cancer or aprecancerous condition in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits conversion of unmethylated H3-K27 to monomethylated H3-K27(H3-K27me1). In one embodiment a method for treating cancer or aprecancerous condition in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits conversion of monomethylated H3-K27 (H3-K27me1) todimethylated H3-K27 (H3-K27me2). In one embodiment a method for treatingcancer or a precancerous condition in a subject comprises administeringto the subject in need thereof a therapeutically effective amount of acompound that inhibits conversion of H3-K27me2 to trimethylated H3-K27(H3-K27me3). In one embodiment a method for treating cancer or aprecancerous condition in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits both conversion of H3-K27me1 to H3-K27me2 and conversionof H3-K27me2 to H3-K27me3. It is important to note that disease-specificincrease in methylation can occur at chromatin in key genomic loci inthe absence of a global increase in cellular levels of histone orprotein methylation. For example, it is possible for aberranthypermethylation at key disease-relevant genes to occur against abackdrop of global histone or protein hypomethylation.

Modulators of methylation can be used for modulating cell proliferation,generally. For example, in some cases excessive proliferation may bereduced with agents that decrease methylation, whereas insufficientproliferation may be stimulated with agents that increase methylation.Accordingly, diseases that may be treated include hyperproliferativediseases, such as benign cell growth and malignant cell growth (cancer).

Exemplary cancers that may be treated include lymphomas, includingnon-Hodgkin lymphoma, follicular lymphoma (FL) and diffuse large B-celllymphoma (DLBCL); melanoma; and leukemia, including CML. Exemplaryprecancerous condition includes myelodisplastic syndrome (MDS; formerlyknown as preleukemia).

Other cancers include Acute Lymphoblastic Leukemia; Acute MyeloidLeukemia; Adrenocortical Carcinoma; AIDS-Related Cancers; AIDS-RelatedLymphoma; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma,Childhood Cerebral; Basal Cell Carcinoma, see Skin Cancer(non-Melanoma); Bile Duct Cancer, Extrahepatic; Bladder Cancer; BoneCancer, osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma;Brain Tumor; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, CerebralAstrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor,Medulloblastoma; Brain Tumor, Supratentorial Primitive NeuroectodermalTumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; BreastCancer; Bronchial Adenomas/Carcinoids; Burkitt's Lymphoma; CarcinoidTumor; Carcinoid Tumor, Gastrointestinal; Carcinoma of Unknown Primary;Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma;Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia;Chronic Myelogenous Leukemia; Chronic Myelogenous Leukemia, Hairy Cell;Chronic Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer;Cutaneous T-Cell Lymphoma, see Mycosis Fungoides and Sezary Syndrome;Endometrial Cancer; Esophageal Cancer; Ewing's Family of Tumors;Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; EyeCancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial; GermCell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; GestationalTrophoblastic Tumor; Glioma; Glioma, Childhood Brain Stem; Glioma,Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway andHypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular(Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer,Childhood (Primary); Hodgkin's Lymphoma; Hodgkin's Lymphoma DuringPregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual PathwayGlioma; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas);Kaposi's Sarcoma; Kidney (Renal Cell) Cancer; Kidney Cancer; LaryngealCancer; Leukemia; Lip and Oral Cavity Cancer; Liver Cancer, Adult(Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-SmallCell; Lung Cancer, Small Cell; Lymphoma, Primary Central Nervous System;Macroglobulinemia, Waldenstrom's; Malignant Fibrous Histiocytoma ofBone/Osteosarcoma; Medulloblastoma; Melanoma; Merkel Cell Carcinoma;Mesothelioma; Mesothelioma, Adult Malignant; Metastatic Squamous NeckCancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome;Multiple Myeloma; Multiple Myeloma/Plasma Cell Neoplasm MycosisFungoides; Myelodysplastic Syndromes; Myelodysplastic/MyeloproliferativeDiseases; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, ChildhoodAcute; Myeloproliferative Disorders, Chronic; Nasal Cavity and ParanasalSinus Cancer; Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin'sLymphoma; Non-Hodgkin's Lymphoma During Pregnancy; Oral Cancer; OralCavity Cancer, Lip and; Oropharyngeal Cancer; Osteosarcoma/MalignantFibrous Histiocytoma of Bone; Ovarian Cancer; Ovarian Epithelial Cancer;Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; PancreaticCancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; ParathyroidCancer; Penile Cancer; Pheochromocytoma; Pineoblastoma andSupratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; PlasmaCell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy andBreast Cancer; Prostate Cancer; Rectal Cancer; Retinoblastoma;Rhabdomyosarcoma; Salivary Gland Cancer; Sarcoma, Ewing's Family ofTumors; Sarcoma, Soft Tissue; Sarcoma, Uterine; Sezary Syndrome; SkinCancer; Skin Cancer (non-Melanoma); Small Intestine Cancer; Soft TissueSarcoma; Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma);Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric)Cancer; Testicular Cancer; Thymoma; Thymoma and Thymic Carcinoma;Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter;Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of;Unusual Cancers of Childhood; Urethral Cancer; Uterine Cancer,Endometrial; Uterine Sarcoma; Vaginal Cancer; Visual Pathway andHypothalamic Glioma; Vulvar Cancer; Waldenstrom's Macroglobulinemia;Wilms' Tumor; and Women's Cancers.

Any other disease in which epigenetic methylation, which is mediated byEZH2, plays a role may be treatable or preventable using compounds andmethods described herein.

For example, neurologic diseases that may be treated include epilepsy,schizophrenia, bipolar disorder or other psychological and/orpsychiatric disorders, neuropathies, skeletal muscle atrophy, andneurodegenerative diseases, e.g., a neurodegenerative disease. Exemplaryneurodegenerative diseases include: Alzheimer's, Amyotrophic LateralSclerosis (ALS), and Parkinson's disease. Another class ofneurodegenerative diseases includes diseases caused at least in part byaggregation of poly-glutamine. Diseases of this class include:Huntington's Diseases, Spinalbulbar Muscular Atrophy (SBMA or Kennedy'sDisease), Dentatorubropallidoluysian Atrophy (DRPLA), SpinocerebellarAtaxia 1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease(MJD; SCA3), Spinocerebellar AtaXia 6 (SCA6), Spinocerebellar Ataxia 7(SCAT), and Spinocerebellar Ataxia 12 (SCAl2).

Combination Therapy

In one aspect of the invention, an EZH2 antagonist, or apharmaceutically acceptable salt thereof, can be used in combinationwith another therapeutic agent to treat diseases such as cancer and/orneurological disorders. For example, the additional agent can be atherapeutic agent that is art-recognized as being useful to treat thedisease or condition being treated by the compound of the presentinvention. The additional agent also can be an agent that imparts abeneficial attribute to the therapeutic composition (e.g., an agent thataffects the viscosity of the composition).

The combination therapy contemplated by the invention includes, forexample, administration of a compound of the invention, or apharmaceutically acceptable salt thereof, and additional agent(s) in asingle pharmaceutical formulation as well as administration of acompound of the invention, or a pharmaceutically acceptable saltthereof, and additional agent(s) in separate pharmaceuticalformulations. In other words, co-administration shall mean theadministration of at least two agents to a subject so as to provide thebeneficial effects of the combination of both agents. For example, theagents may be administered simultaneously or sequentially over a periodof time.

The agents set forth below are for illustrative purposes and notintended to be limiting. The combinations, which are part of thisinvention, can be the compounds of the present invention and at leastone additional agent selected from the lists below. The combination canalso include more than one additional agent, e.g., two or threeadditional agents if the combination is such that the formed compositioncan perform its intended function.

For example, one aspect of the invention relates to the use of an EZH2antagonist in combination with another agent for the treatment of cancerand/or a neurological disorder. In one embodiment, an additional agentis an anticancer agent that is a compound that affects histonemodifications, such as an HDAC inhibitor. In certain embodiments, anadditional anticancer agent is selected from the group consisting ofchemotherapetics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG, Abraxane™,Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-trans retinoic acid,amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®, Camptosar®, CeeNU®,Clofarabine, Clolar™, Cytoxan®, daunorubicin hydrochloride, DaunoXome®,Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate,Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®,Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixempra®, L-asparaginase,Leukeran®, liposomal Ara-C, L-PAM, Lysodren, Matulane®, mithracin,Mitomycin-C, Myleran®, Navelbine®, Neutrexin®, nilotinib, Nipent®,Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®, Paraplatin®,Platinol®, prolifeprospan 20 with carmustine implant, Sandostatin®,Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA, Trisenox®, Valstar®,Velban®, Vidaza™, vincristine sulfate, VM 26, Xeloda® and Zanosar®);biologics (such as Alpha Interferon, Bacillus Calmette-Guerin, Bexxar®,Campath®, Ergamisol®, Erlotinib, Herceptin®, Interleukin-2, Iressa®,lenalidomide, Mylotarg®, Ontak®, Pegasys®, Revlimid®, Rituxan®,Tarceva™, Thalomid®, Velcade® and Zevalin™); small molecules (such asTykerb®); corticosteroids (such as dexamethasone sodium phosphate,DeltaSone® and Delta-Cortef®); hormonal therapies (such as Arimidex®,Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®,Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, Plenaxis™ andZoladex®); and radiopharmaceuticals (such as Iodotope®, Metastron®,Phosphocol® and Samarium SM-153).

Dosage

As used herein, a “therapeutically effective amount” or “therapeuticallyeffective dose” is an amount of an EZH2 antagonist or a combination oftwo or more such compounds, which inhibits, totally or partially, theprogression of the condition or alleviates, at least partially, one ormore symptoms of the condition. A therapeutically effective amount canalso be an amount which is prophylactically effective. The amount whichis therapeutically effective will depend upon the patient's size andgender, the condition to be treated, the severity of the condition andthe result sought. In one embodiment, a therapeutically effective doserefers to that amount of the EZH2 antagonists that result inamelioration of symptoms in a patient. For a given patient, atherapeutically effective amount may be determined by methods known tothose of skill in the art.

Toxicity and therapeutic efficacy of EZH2 antagonists can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the maximum tolerated dose (MTD) and theED₅₀ (effective dose for 50% maximal response). The dose ratio betweentoxic and therapeutic effects is the therapeutic index and it can beexpressed as the ratio between MTD and ED₅₀. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in humans. Dosage may also be guided bymonitoring the EZH2 antagonist's effect on pharmacodynamic markers ofenzyme inhibition (e.g., histone methylation or target gene expression)in diseased or surrogate tissue. Cell culture or animal experiments canbe used to determine the relationship between doses required for changesin pharmacodynamic markers and doses required for therapeutic efficacycan be determined in cell culture or animal experiments or early stageclinical trials. The dosage of such EZH2 antagonists lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. In thetreatment of crises, the administration of an acute bolus or an infusionapproaching the MTD may be required to obtain a rapid response.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themethyltransferase modulating effects, or minimal effective concentration(MEC) for the required period of time to achieve therapeutic efficacy.The MEC will vary for each EZH2 antagonist but can be estimated from invitro data and animal experiments. Dosages necessary to achieve the MECwill depend on individual characteristics and route of administration.However, high pressure liquid chromatography (HPLC) assays or bioassayscan be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value. In certainembodiments, EZH2 antagonists should be administered using a regimenwhich maintains plasma levels above the MEC for 10-90% of the time,preferably between 30-90% and most preferably between 50-90% until thedesired amelioration of symptoms is achieved. In other embodiments,different MEC plasma levels will be maintained for differing amounts oftime. In cases of local administration or selective uptake, theeffective local concentration of the drug may not be related to plasmaconcentration.

One of skill in the art can select from a variety of administrationregimens and the amount of EZH2 antagonist administered will, of course,be dependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgment of the prescribing physician.

Compounds and Pharmaceutical Compositions

Aspects of the invention concern compounds which are useful according tothe methods of the invention. These compounds are referred to herein as“inhibitors of EZH2” and, equivalently, “EZH2 antagonists”. Thecompounds can be presented as the compounds per se, pharmaceuticallyacceptable salts of the compounds, or as pharmaceutical compositions.

The compounds suitable for use in the method of this invention includecompounds of Formula (I):

wherein,

V¹ is N or CR⁷,

V² is N or CR², provided when. V¹ is N, V² is N,

X and Z are selected independently from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, unsubstituted orsubstituted (C₃-C₈)cycloalkyl, unsubstituted or substituted(C₃-C₈)cycloalkyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted (C₅-C₈)cycloalkenyl, unsubstituted or substituted(C₅-C₈)cycloalkenyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl,(C₆-C₁₀)bicycloalkyl, unsubstituted or substituted heterocycloalkyl,unsubstituted or substituted heterocycloalkyl-(C₁-C₈)alkyl or—(C₂-C₈)alkenyl, unsubstituted or substituted aryl, unsubstituted orsubstituted aryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted heteroaryl, unsubstituted or substitutedheteroaryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, halo, cyano, —COR^(a),—CO₂R^(a), —CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b), —SR^(a), —SOR^(a),—SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),—NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),—NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—NR^(a)NR^(a)C(O)NR^(a)R^(b), —NR^(a)NR^(a)C(O)ORa, —OR^(a),—OC(O)R^(a), and —OC(O)NR^(a)R^(b);

Y is H or halo;

R¹ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, unsubstituted orsubstituted (C₃-C₈)cycloalkyl, unsubstituted or substituted(C₃-C₈)cycloalkyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted (C₅-C₈)cycloalkenyl, unsubstituted or substituted(C₅-C₈)cycloalkenyl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, unsubstituted orsubstituted (C₆-C₁₀)bicycloalkyl, unsubstituted or substitutedheterocycloalkyl or —(C₂-C₈)alkenyl, unsubstituted or substitutedheterocycloalkyl-(C₁-C₈)alkyl, unsubstituted or substituted aryl,unsubstituted or substituted aryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl,unsubstituted or substituted heteroaryl, unsubstituted or substitutedheteroaryl-(C₁-C₈)alkyl or —(C₂-C₈)alkenyl, —COR^(a), —CO₂R^(a),—CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b);

R² is hydrogen, (C₁-C₈)alkyl, trifluoromethyl, alkoxy, or halo, in whichsaid (C₁-C₈)alkyl is optionally substituted with one to two groupsselected from amino and (C₁-C₃)alkylamino;

R⁷ is hydrogen, (C₁-C₃)alkyl, or alkoxy;

R³ is hydrogen, (C₁-C₈)alkyl, cyano, trifluoromethyl, —NR^(a)R^(b), orhalo;

R⁶ is selected from the group consisting of hydrogen, halo,(C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, unsubstituted orsubstituted (C₃-C₈)cycloalkyl, unsubstituted or substituted(C₃-C₈)cycloalkyl-(C₁-C₈)alkyl, unsubstituted or substituted(C₅-C₈)cycloalkenyl, unsubstituted or substituted(C₅-C₈)cycloalkenyl-(C₁-C₈)alkyl, (C₆-C₁₀)bicycloalkyl, unsubstituted orsubstituted heterocycloalkyl, unsubstituted or substitutedheterocycloalkyl-(C₁-C₈)alkyl, unsubstituted or substituted aryl,unsubstituted or substituted aryl-(C₁-C₈)alkyl, unsubstituted orsubstituted heteroaryl, unsubstituted or substitutedheteroaryl-(C₁-C₈)alkyl, cyano, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b),—CONR^(a)NR^(a)R^(b), —SR^(a), —SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b),nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b),—NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b),—NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—NR^(a)NR^(a)C(O)NR^(a)R^(b), —NR^(a)NR^(a)C(O)OR^(a), —OR^(a),—OC(O)R^(a), —OC(O)NR^(a)R^(b);

wherein any (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, cycloalkyl,cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl, or heteroaryl groupis optionally substituted by 1, 2 or 3 groups independently selectedfrom the group consisting of —O(C₁-C₆)alkyl(R^(c))₁₋₂,—S(C₁-C₆)alkyl(R^(c))₁₋₂, —(C₁-C₆)alkyl(R^(c))₁₋₂,—(C₁-C₈)alkyl-heterocycloalkyl, (C₃-C₈)cycloalkyl-heterocycloalkyl,halo, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,(C₁-C₆)haloalkyl, cyano, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —SR^(a),—SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b),—NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a),—NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a),OC(O)NR^(a)R^(b), heterocycloalkyl, aryl, heteroaryl, aryl(C₁-C₄)alkyl,and heteroaryl(C₁-C₄)alkyl;

-   -   wherein any aryl or heteroaryl moiety of said aryl, heteroaryl,        aryl(C₁-C₄)alkyl, or heteroaryl(C₁-C₄)alkyl is optionally        substituted by 1, 2 or 3 groups independently selected from the        group consisting of halo, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano, —COR^(a),        —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), —SOR^(a), —SO₂R^(a),        —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),        —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),        —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a), and        —OC(O)NR^(a)R^(b);

R^(a) and R^(b) are each independently hydrogen, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,(C₆-C₁₀)bicycloalkyl, heterocycloalkyl, aryl, or heteroaryl, whereinsaid (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, cycloalkyl,cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl or heteroaryl groupis optionally substituted by 1, 2 or 3 groups independently selectedfrom halo, hydroxyl, (C₁-C₄)alkoxy, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, —CO₂H, —CO₂(C₁-C₄)alkyl, —CONH₂,—CONH(C₁-C₄)alkyl,

—CON((C₁-C₄)alkyl)((C₁-C₄)alkyl), —SO₂(C₁-C₄)alkyl, —SO₂NH₂,—SO₂NH(C₁-C₄)alkyl, and SO₂N((C₁-C₄)alkyl)((C₁-C₄)alkyl);

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 5-8 membered saturated or unsaturated ring,optionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein said ring is optionally substituted by 1,2 or 3 groups independently selected from (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyeamino, hydroxyl, oxo, (C₁-C₄)alkoxy, and(C₁-C₄)alkoxy(C₁-C₄)alkyl, wherein said ring is optionally fused to a(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring;

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 6- to 10-membered bridged bicyclic ring systemoptionally fused to a (C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, orheteroaryl ring;

each R^(c) is independently (C₁-C₄)alkylamino, —NR^(a)SO2R^(b),—SOR^(a), —SO₂R^(a), —NR^(a)C(O)OR^(a), —NR^(a)R^(b), or —CO₂R^(a);

or a salt thereof.

Subgroups of the compounds encompassed by the general structure ofFormula (I) are represented as follows:

Subgroup A of Formula (I)

X and Z are selected from the group consisting of (C₁-C₈)alkyl,(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —NR^(a)R^(b), and—OR^(a);

Y is H or F;

R¹ is selected from the group consisting of (C₁-C₈)alkyl,(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, and heteroaryl;

R² is hydrogen, (C₁-C₈)alkyl, trifluoromethyl, alkoxy, or halo, in whichsaid (C₁-C₈)alkyl is optionally substituted with one to two groupsselected from amino and (C₁-C₃)alkylamino;

R⁷ is hydrogen, (C₁-C₃)alkyl, or alkoxy;

R³ is selected from the group consisting of hydrogen, (C₁-C₈)alkyl,cyano, trifluoromethyl, —NR^(a)R^(b), and halo;

R⁶ is selected from the group consisting of hydrogen, halo, cyano,trifluoromethyl, amino, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl; aryl,heteroaryl, acylamino; (C₂-C₈)alkynyl, arylalkynyl, heteroarylalkynyl;—SO₂R^(a); —SO₂NR^(a)R^(b) and —NR^(a)SO₂R^(b);

-   -   wherein any (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₈)alkynyl,        arylalkynyl, heteroarylalkynyl group is optionally substituted        by 1, 2 or 3 groups independently selected from        —O(C₁-C₆)alkyl(R^(c))₁₋₂, —S(C₁-C₆)alkyl(R^(c))₁₋₂,        —(C₁-C₆)alkyl(R^(c))₁₋₂, —(C₁-C₈)alkyl-heterocycloalkyl,        (C₃-C₈)cycloalkyl-heterocycloalkyl, halo, (C₁-C₆)alkyl,        (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano,        —COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), —SOR^(a),        —SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b),        —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a),        —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a),        —OC(O)NR^(a)R^(b), heterocycloalkyl, aryl, heteroaryl,        aryl(C₁-C₄)alkyl, and heteroaryl(C₁-C₄)alkyl;

R^(a) and R^(b) are each independently hydrogen, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,(C₆-C₁₀)bicycloalkyl, heterocycloalkyl, aryl, or heteroaryl, whereinsaid (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, cycloalkyl,cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl or heteroaryl groupis optionally substituted by 1, 2 or 3 groups independently selectedfrom halo, hydroxyl, (C₁-C₄)alkoxy, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, —CO₂H, —CO₂(C₁-C₄)alkyl, —CONH₂,—CONH(C₁-C₄)alkyl, —CON((C₁-C₄)alkyl)((C₁-C₄)alkyl), —SO₂(C₁-C₄)alkyl,—SO₂NH₂, —SO₂NH(C₁-C₄)alkyl, and —SO₂N((C₁-C₄)alkyl)((C₁-C₄)alkyl);

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 5-8 membered saturated or unsaturated ring,optionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein said ring is optionally substituted by 1,2 or 3 groups independently selected from (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, hydroxyl, oxo, (C₁-C₄)alkoxy, and(C₁-C₄)alkoxy(C₁-C₄)alkyl, wherein said ring is optionally fused to a(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring;

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 6- to 10-membered bridged bicyclic ring systemoptionally fused to a (C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, orheteroaryl ring. An aryl or heteroaryl group in this particular subgroupA is selected independently from the group consisting of furan,thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, oxadiazole,thiadiazole, triazole, tetrazole, benzofuran, benzothiophene,benzoxazole, benzothiazole, phenyl, pyridine, pyridazine, pyrimidine,pyrazine, triazine, tetrazine, quinoline, cinnoline, quinazoline,quinoxaline, and naphthyridine or another aryl or heteroaryl group asfollows:

wherein in (1),

A is O, NH, or S; B is CH or N, and C is hydrogen or C₁-C₈ alkyl; or

wherein in (2),

D is N or C optionally substituted by hydrogen or C₁-C₈ alkyl; or

wherein in (3),

E is NH or CH₂; F is O or CO; and G is NH or CH₂; or

wherein in (4),

J is O, S or CO; or

wherein in (5),

Q is CH or N;

M is CH or N; and

L/(5) is hydrogen, halo, amino, cyano, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,—COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)SO₂R^(b),—NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—NR^(a)NR^(a)C(O)NR^(a)R^(b), or —OR^(a),

-   -   wherein any (C₁-C₈)alkyl or (C₃-C₈)cycloalkyl group is        optionally substituted by 1, 2 or 3 groups independently        selected from (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano, —COR^(a),        —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), —SOR^(a), —SO₂R^(a),        —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),        —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),        —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a), and        —OC(O)NR^(a)R^(b); wherein R^(a) and R^(b) are defined as above;        or

wherein in (6),

L/(6) is NH or CH₂; or

wherein in 7,

-   -   M/(7) is hydrogen, halo, amino, cyano, (C₁-C₈)alkyl,        (C₃-C₈)cycloalkyl, heterocycloalkyl, —COR^(a), —CO₂R^(a),        —CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b), —SO₂R^(a),        —SO₂NR^(a)R^(b), —NR^(a)R^(b), —NR^(a)C(O)R^(b),        —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b),        —NR^(a)NR^(a)C(O)R^(b), —NR^(a)NR^(a)C(O)NR^(a)R^(b), or        —OR^(a),    -   wherein any (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, or heterocycloalkyl        group is optionally substituted by 1, 2 or 3 groups        independently selected from (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano, —COR^(a),        —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), —SOR^(a), —SO₂R^(a),        —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),        —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),        —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a), and        —OC(O)NR^(a)R^(b); wherein R^(a) and R^(b) are defined as above;        or

wherein in (8),

P is CH₂, NH, O, or S; Q/(8) is CH or N; and n is 0-2; or

wherein in (9),

S/(9) and T/(9) is C, or S/(9) is C and T/(9) is N, or S/(9) is N andT/(9) is C;

R is hydrogen, amino, methyl, trifluoromethyl, or halo;

U is hydrogen, halo, amino, cyano, nitro, trifluoromethyl, (C₁-C₈)alkyl,(C₃-C₈)cycloalkyl, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)SO₂R^(b),

—NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—OR^(a), or 4-(1H-pyrazol-4-yl),

-   -   wherein any (C₁-C₈)alkyl or (C₃-C₈)cycloalkyl group is        optionally substituted by 1, 2 or 3 groups independently        selected from (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano, —COR^(a),        —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), SOR^(a), —SO₂R^(a),        —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),        —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),        —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a), and        —OC(O)NR^(a)R^(b); wherein R^(a) and R^(b) are defined as above.

Subgroup B of Formula (I)

X and Z are selected independently from the group consisting of(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, heteroaryl,—NR^(a)R^(b), and —OR^(a);

Y is H;

R¹ is (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, or heterocycloalkyl;

R² is hydrogen, (C₁-C₃)alkyl, or halo, in which said (C₁-C₃)alkyl isoptionally substituted with one to two groups selected from amino and(C₁-C₃)alkylamino;

R⁷ is hydrogen, (C₁-C₃)alkyl, or alkoxy;

R³ is hydrogen, (C₁-C₈)alkyl or halo;

R⁶ is hydrogen, halo, cyano, trifluoromethyl, amino, (C₁-C₈)alkyl,(C₃-C₈)cycloalkyl, aryl, heteroaryl, acylamino; (C₂-C₈)alkynyl,arylalkynyl, heteroarylalkynyl, —SO₂R^(a), —SO₂NR^(a)R^(b), or

—NR^(a)SO₂R^(b);

-   -   wherein any (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₈)alkynyl,        arylalkynyl, or heteroarylalkynyl group is optionally        substituted by 1, 2 or 3 groups independently selected from        halo, (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,        (C₁-C₆)haloalkyl, cyano, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b),        —SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b),        —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a),        —NR^(a)SO₂R^(b), NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a),        —OC(O)NR^(a)R^(b), heterocycloalkyl, aryl, heteroaryl,        aryl(C₁-C₄)alkyl, and heteroaryl(C₁-C₄)alkyl;

R^(a) and R^(b) are each independently hydrogen, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,(C₆-C₁₀)bicycloalkyl, heterocycloalkyl, aryl, or heteroaryl, whereinsaid (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, cycloalkyl,cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl or heteroaryl groupis optionally substituted by 1, 2 or 3 groups independently selectedfrom halo, hydroxyl, (C₁-C₄)alkoxy, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, —CO₂H, —CO₂(C₁-C₄)alkyl, —CONH₂,—CONH(C₁-C₄)alkyl,

—CON((C₁-C₄)alkyl)((C₁-C₄)alkyl), —SO₂(C₁-C₄)alkyl, —SO₂NH₂,—SO₂NH(C₁-C₄)alkyl, and —SO₂N((C₁-C₄)alkyl)((C₁-C₄)alkyl);

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 5-8 membered saturated or unsaturated ring,optionally containing an additional heteroatom selected from oxygen,nitrogen, and sulfur, wherein said ring is optionally substituted by 1,2 or 3 groups independently selected from (C₁-C₄)alkyl,(C₁-C₄)haloalkyl, amino, (C₁-C₄)alkylamino,((C₁-C₄)alkyl)((C₁-C₄)alkyl)amino, hydroxyl, oxo, (C₁-C₄)alkoxy, and(C₁-C₄)alkoxy(C₁-C₄)alkyl, wherein said ring is optionally fused to a(C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring;

or R^(a) and R^(b) taken together with the nitrogen to which they areattached represent a 6- to 10-membered bridged bicyclic ring systemoptionally fused to a (C₃-C₈)cycloalkyl, heterocycloalkyl, aryl, orheteroaryl ring. Aryl and heteroaryl in this definition are selectedfrom the group consisting of furan, thiophene, pyrrole, oxazole,thiazole, imidazole, pyrazole, oxadiazole, thiadiazole, triazole,tetrazole, benzofuran, benzothiophene, benzoxazole, benzothiazole,phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, tetrazine,quinoline, cinnoline, quinazoline, quinoxaline, and naphthyridine or acompound of another aryl or heteroaryl group as follows:

wherein in (1),

A is O, NH, or S; B is CH or N, and C is hydrogen or C₁-C₈ alkyl; or

wherein in (2),

D is N or C optionally substituted by hydrogen or C₁-C₈ alkyl; or

wherein in (3),

E is NH or CH₂; F is O or CO; and G is NH or CH₂; or

wherein in (4),

J is O, S or CO; or

wherein in (5),

Q is CH or N;

M is CH or N; and

L/(5) is hydrogen, halo, amino, cyano, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,—COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)SO₂R^(b),—NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)(O)R^(b),—NR^(a)NR^(a)C(O)NR^(a)R^(b), or —OR^(a),

-   -   wherein any (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, group is optionally        substituted by 1, 2 or 3 groups independently selected from        (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl, (C₅-C₈)cycloalkenyl,        (C₁-C₆)haloalkyl, cyano, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b),        —SR^(a), —SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b), nitro,        —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b),        —NR^(a)C(O)OR^(a), NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b),        —OR^(a), —OC(O)R^(a), and —OC(O)NR^(a)R^(b),        wherein R^(a) and R^(b) are defined as above; or

wherein in (6),

L/(6) is NH or CH₂; or

wherein in (7),

-   -   M/(7) is hydrogen, halo, amino, cyano, (C₁-C₈)alkyl,        (C₃-C₈)cycloalkyl, heterocycloalkyl, —COR^(a), —CO₂R^(a),        —CONR^(a)R^(b), —CONR^(a)NR^(a)R^(b), —SO₂R^(a),        —SO₂NR^(a)R^(b), —NR^(a)R^(b), —NR^(a)C(O)R^(b),        —NR^(a)SO₂R^(b), —NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b),        —NR^(a)NR^(a)C(O)R^(b), —NR^(a)NR^(a)C(O)NR^(a)R^(b), or        —OR^(a),    -   wherein any (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, heterocycloalkyl        group is optionally substituted by 1, 2 or 3 groups        independently selected from (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano, —COR^(a),        —CO₂R^(a), —CONR^(a)R^(b), —SR^(a), —SOR^(a), —SO₂R^(a),        —SO₂NR^(a)R^(b), nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b),        NR^(a)C(O)NR^(a)R^(b), —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b),        —NR^(a)SO₂NR^(a)R^(b), —OR^(a), —OC(O)R^(a), —OC(O)NR^(a)R^(b);        wherein R^(a) and R^(b) are defined as above; or

wherein in (8),

P is CH₂, NH, O, or S; Q/(8) is CH or N; and n is 0-2; or

wherein in (9),

S/(9) and T/(9) is C, or S/(9) is C and T/(9) is N, or S/(9) is N andT/(9) is C;

R is hydrogen, amino, methyl, trifluoromethyl, halo;

U is hydrogen, halo, amino, cyano, nitro, trifluoromethyl, (C₁-C₈)alkyl,(C₃-C₈)cycloalkyl, —COR^(a), —CO₂R^(a), —CONR^(a)R^(b), —SO₂R^(a),—SO₂NR^(a)R^(b), —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)SO₂R^(b),

—NR^(a)SO₂NR^(a)R^(b), —NR^(a)NR^(a)R^(b), —NR^(a)NR^(a)C(O)R^(b),—OR^(a), or 4-(1H-pyrazol-4-yl),

-   -   wherein any (C_(j)—C₈)alkyl, or (C₃-C₈)cycloalkyl group is        optionally substituted by 1, 2 or 3 groups independently        selected from (C₁-C₆)alkyl, (C₃-C₈)cycloalkyl,        (C₅-C₈)cycloalkenyl, (C₁-C₆)haloalkyl, cyano, —COR^(a),        —CO₂R^(a), —CONR^(a)R^(b), —SOR^(a), —SO₂R^(a), —SO₂NR^(a)R^(b),        nitro, —NR^(a)R^(b), —NR^(a)C(O)R^(b), —NR^(a)C(O)NR^(a)R^(b),        —NR^(a)C(O)OR^(a), —NR^(a)SO₂R^(b), —NR^(a)SO2NR^(a)R^(b),        —OR^(a), —OC(O)R^(a), and —OC(O)NR^(a)R^(b), wherein R^(a) and        R^(b) are defined as above.

Subgroup C of Formula (I)

X is methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl, trifluoromethyl, tetrahydropyran,hydroxymethyl, methoxymethyl, or benzyl;

Y is H;

Z is methyl, ethyl, n-propyl, isopropyl, trifluoromethyl, or benzyl;

R¹ is isopropyl, tert-butyl, cyclobutyl, cyclopentyl, cyclohexyl,(1-methylethyl)cyclopropyl, 1,1-dioxo-tetrahydrothiophene-3-yl,1-Me-piperidin-4-yl, tetrahydrofuran-3-yl, tetrahydropyran-4-yl,N,N-dimethyl-1-propanaminyl, benzyl, or 4-pyridyl;

R² is hydrogen, (C₁-C₃)alkyl, or halo, in which said (C₁-C₃)alkyl isoptionally substituted with one to two groups selected from amino and(C₁-C₃)alkylamino;

R⁷ is hydrogen, (C₁-C₃)alkyl, or alkoxy;

R³ is H, methyl, or Br; and

R⁶ is methyl, bis(1,1-dimethylethyl), bis(1-methylethyl), cyclopropyl,propyl, dimethylamino, ethylamino, (2-hydroxyethyl)amino,2-propen-1-ylamino, 1-piperazinyl, 1-piperidinyl, 4-morpholinyl,4-piperidinylamino, tetrahydro-2H-pyran-4-ylamino, phenylamino,(phenylmethyl)amino, (4-pyridinylmethyl)amino,[2-(2-pyridinylamino)ethyl]amino, 2-(dimethylamino)ethyl]amino,4-pyridinylamino, 4-(aminocarbonyl)phenyl]amino,3-hydroxy-3-methyl-1-butyn-1-yl, 4-pyridinylethynyl, phenylethynyl,2-furanyl, 3-thienyl; 1H-pyrazol-4-yl, 1H-pyrazol-5-yl, 1H-indazol-6-yl,3-methyl-1H-indazol-5-yl, 1H-1,2,3-benzotriazol-5-yl,2-oxo-2,3-dihydro-1H-benzimidazol-5-yl, 2-oxo-2,3-dihydro-1H-indol-5-yl,2-oxo-2,3-dihydro-1H-indol-6-yl, 2,1,3-benzoxadiazol-5-yl,2-amino-6-quinazolinyl, 2,4-dioxo-1,2,3,4-tetrahydro-5-pyrimidinyl,2-amino-5-pyrimidinyl, 7-oxo-1,5,6,7-tetrahydro-1,8-naphthyridin-3-yl,phenyl, 2-methylphenyl, 2-nitrophenyl, 2-phenylethyl, 3-aminophenyl,4-aminophenyl, 4-chlorophenyl, 4-fluorophenyl, 4-(methyloxy)phenyl,3-(acetylamino)phenyl, 4-(acetylamino)phenyl, 4-(aminocarbonyl)phenyl,4-(1H-pyrazol-4-yl)phenyl, 4-(aminosulfonyl)phenyl,4-(methylsulfonyl)phenyl, 4-[(dimethylamino)sulfonyl]phenyl,4-[(methylamino)carbonyl]phenyl, 4-[(methylamino)sulfonyl]phenyl,4-[(methylsulfonyl)amino]phenyl, 3-pyridinyl, 4-pyridinyl,2-(4-morpholinyl)-4-pyridinyl, 2-amino-4-pyridinyl,5-(methyloxy)-3-pyridinyl, 5-(methylsulfonyl)-3-pyridinyl,5-[(cyclopropylsulfonyl)amino]-6-(methyloxy)-3-pyridinyl,5-[(phenylsulfonyl)amino]-3-pyridinyl,6-(4-methyl-1-piperazinyl)-3-pyridinyl, 6-(4-morpholinyl)-3-pyridinyl,6-(acetylamino)-3-pyridinyl, 6-(dimethylamino)-3-pyridinyl,6-(methyloxy)-3-pyridinyl, 6-[(methylamino)carbonyl]-3-pyridinyl,6-Rmethylamino)sulfonyl]-3-pyridinyl, 6-methyl-3-pyridinyl, or4-pyridinyloxy. (See, e.g., WO 2011/140325; WO 2011/140324; and WO2012/005805, each of which is incorporated by reference in itsentirety.)

The compounds suitable for use in the method of this invention alsoinclude compounds of Formula (II):

wherein

X₁ is N or CR₁₁;

X₂ is N or CR₁₃;

Z₁ is NR₇R₈, OR_(S), SR_(S), or CR₇R₈R₁₄;

each of R₁, R₅, R₉, and R₁₀, independently, is H or C₁-C₆ alkyloptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl;

each of R₂, R₃, and R₄, independently, is -Q₁-T₁, in which Q₁ is a bondor C₁-C₃ alkyl linker optionally substituted with halo, cyano, hydroxylor C₁-C₆ alkoxy, and T₁ is H, halo, hydroxyl, COOH, cyano, or R₅₁, inwhich R_(S), is C₁-C₃ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxyl, C(O)O—C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and R_(S1) isoptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, oxo, COOH, C(O)O—C₁-C₆ alkyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl;

R₆ is C₆-C₁₀ aryl or 5- or 6-membered heteroaryl, each of which isoptionally substituted with one or more -Q₂-T₂, wherein Q₂ is a bond orC₁-C₃ alkyl linker optionally substituted with halo, cyano, hydroxyl orC₁-C₆ alkoxy, and T₂ is H, halo, cyano, —OR_(a), —NR_(a)R_(b),—(NR_(a)R_(b)R_(c))⁺A⁻, —C(O)R_(a), —C(O)OR_(a), —C(O)NR_(a)R_(b),—NR_(b)C(O)R_(a), —NR_(b)C(O)OR_(a), —S(O)₂R_(a), —S(O)₂NR_(a)R_(b), orR_(S2), in which each of R_(a), R_(b), and R_(c), independently is H orR_(S3), A⁻ is a pharmaceutically acceptable anion, each of R_(S2) andR_(S3), independently, is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, orR_(a) and R_(b), together with the N atom to which they are attached,form a 4 to 12-membered heterocycloalkyl ring having 0 or 1 additionalheteroatom, and each of R_(S2), R_(S3), and the 4 to 12-memberedheterocycloalkyl ring formed by R_(a) and R_(b), is optionallysubstituted with one or more -Q₃-T₃, wherein Q₃ is a bond or C₁-C₃ alkyllinker each optionally substituted with halo, cyano, hydroxyl or C₁-C₆alkoxy, and T₃ is selected from the group consisting of halo, cyano,C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, 5- or 6-membered heteroaryl, OR_(d), COOR_(d),—S(O)₂R_(d), —NR_(d)R_(e), and —C(O)NR_(d)R_(e), each of R_(d) and R_(e)independently being H or C₁-C₆ alkyl, or -Q₃-T₃ is oxo; or any twoneighboring -Q₂-T₂, together with the atoms to which they are attachedform a 5- or 6-membered ring optionally containing 1-4 heteroatomsselected from N, O and S and optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl, COOH,C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl; provided that -Q₂-T₂is not H;

R₇ is -Q₄-T₄, in which Q₄ is a bond, C₁-C₄ alkyl linker, or C₂-C₄alkenyl linker, each linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₄ is H, halo, cyano, NR_(f)R_(g),—OR_(f), —C(O)R_(f), —C(O)OR_(f), —C(O)NR_(f)R_(g), —C(O)NR_(f)OR_(g),—NR_(f)C(O)R_(g), —S(O)₂R_(f), or R_(S4), in which each of R_(f) andR_(g), independently is H or R_(S5), each of R_(S4) and R_(S5),independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl, and each of R_(S4) and R_(S5) is optionallysubstituted with one or more -Q₅-T₅, wherein Q is a bond, C(O),C(O)NR_(k), NR_(k)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(k) being H orC₁-C₆ alkyl, and T₅ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(q)R_(q) in which q is 0, 1, or 2 andR_(q) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₅ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₅ is H,halo, hydroxyl, or cyano; or -Q₅-T₅ is oxo; provided that R₇ is not H;

each of R₈, R₁₁, R₁₂, and R₁₃, independently, is H, halo, hydroxyl,COOH, cyano, R_(S6), OR_(S6), or COOR_(S6), in which R_(S6) is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, 4 to 12-memberedheterocycloalkyl, amino, mono-C₁-C₆ alkylamino, or di-C₁-C₆ alkylamino,and R_(S6) is optionally substituted with one or more substituentsselected from the group consisting of halo, hydroxyl, COOH, C(O)O—C₁-C₆alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆alkylamino; or R₇ and R₈, together with the N atom to which they areattached, form a 4 to 11-membered heterocycloalkyl ring having 0 to 2additional heteroatoms, or R₇ and R₈, together with the C atom to whichthey are attached, form C₃-C₈ cycloalkyl or a 4 to 11-memberedheterocycloalkyl ring having 1 to 3 heteroatoms, and each of the 4 to11-membered heterocycloalkyl rings or C₃-C₈ cycloalkyl formed by R₇ andR₅ is optionally substituted with one or more -Q₆-T₆, wherein Q6 is abond, C(O), C(O)NR_(m), NR_(m)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(m)being H or C₁-C₆ alkyl, and T₆ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(p)R_(p) in which p is 0, 1, or 2 andR_(p) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₆ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₆ is H,halo, hydroxyl, or cyano; or -Q₆-T₆ is oxo; and

R₁₄ is absent, H, or C₁-C₆ alkyl optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl, COOH,C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl.

One subset of the compounds of Formula (II) includes those of Formula(Ha):

Another subset of the compounds of Formula (II) includes those ofFormula (IIb), (IIc), or (IId):

The compounds of Formulae (II), (IIa), (IIc), and (IId) can include oneor more of the following features:

For example, X₁ is CR₁₁ and X₂ is CR₁₃.

For example, X₁ is CR₁₁ and X₂ is N.

For example, X₁ is N and X₂ is CR₁₃.

For example, X₁ is N and X₂ is N.

For example, Z₁ is NR₇R₈.

For example, Z₁ is CR₇R₈R₁₄.

For example, Z₁ is OR₇.

For example, Z₁ is SR₇,

For example, R₆ is phenyl substituted with one or more -Q₂-T₂.

For example, R₆ is 5 to 6-membered heteroaryl containing 1-3 additionalheteroatoms selected from N, O, and S and optionally substituted withone or more -Q₂-T₂.

For example, R₆ is pyridinyl, pyrazolyl, pyrimidinyl, quinolinyl,tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, furyl, orthienyl, each of which is optionally substituted with one or more-Q₂-T₂.

For example, Q₂ is a bond.

For example, Q2 is an unsubstituted C₁-C₃ alkyl linker.

For example, T₂ is C₁-C₆ alkyl or C₆-C₁₀ aryl, each optionallysubstituted with one or more -Q₃-T₃.

For example, T₂ is an unsubstituted substituted straight chain C₁-C₆ orbranched C₃-C₆ alkyl, including but not limited to, methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl andn-hexyl.

For example, T₂ is phenyl.

For example, T₂ is halo (e.g., fluorine, chlorine, bromine, and iodine).

For example, T₂ is 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, tetrahydro-2H-thiopyran,morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like) optionally substituted with one or more -Q₃-T₃.

For example, T₂ is —OR_(a), —NR_(a)R_(b), —(NR_(a)R_(b)R_(c))⁺A⁻,—C(O)R_(a), —C(O)OR_(a), —C(O)NR_(a)R_(b), —NR_(b)C(O)R_(a),—NR_(b)C(O)OR_(a), —S(O)₂R_(a), or —S(O)₂NR_(a)R_(b).

For example, T₂ is —NR_(a)R_(b) or —C(O)NR_(a)R_(b), in which each ofR_(a) and R_(b), independently is H or C₁-C₆ alkyl, or R_(a) and R_(b),together with the N atom to which they are attached, form a 4 to7-membered heterocycloalkyl ring having 0 or 1 additional heteroatom,the C₁-C₆ alkyl and the 4 to 7-membered heterocycloalkyl ring beingoptionally substituted with one or more -Q₃-T₃.

For example, Q₂ is C₁-C₃ alkyl linker optionally substituted with haloor hydroxyl.

For example, Q₂ is a bond or methyl linker and T₂ is H, halo, —OR_(a),—NR_(a)R_(b), —(NR_(a)R_(b)R_(c))⁺A⁻, or —S(O)₂NR_(a)R_(b).

For example, each of R_(a), R_(b), and R_(e), independently is H orC₁-C₆ alkyl optionally substituted with one or more -Q₃-T₃.

For example, one of R_(a), R_(b), and R_(c) is H.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms to the N atom (e.g., azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like) and the ring is optionally substituted with one or more-Q₃-T₃.

For example, -Q₃-T₃ is oxo.

For example, T₂ is 4 to 7-membered heterocycloalkyl or C₃-C₈ cycloalkyland one or more -Q₃-T₃ are oxo.

For example, Q₃ is a bond or unsubstituted or substituted C₁-C₃ alkyllinker.

For example, T₃ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃alkyl, OR_(d), COOR_(d), —S(O)₂R_(d), or —NR_(d)R_(e).

For example, one of R_(d) and R_(e) is H.

For example, R₇ is —C(O)R_(f).

For example, R₇ is —C(O)R_(f), in which R_(f) is C₃-C₈ cycloalkyl.

For example, R₇ is C₆-C₁₀ aryl substituted with one or more -Q₅-T₅.

For example, R₇ is phenyl optionally substituted with one or more-Q₅-T₅.

For example, R₇ is C₁-C₆ alkyl optionally substituted with one or more-Q₅-T₅.

For example, R₇ is C₃-C₈ cycloalkyl optionally substituted with one ormore -Q₅-T₅.

For example, R₇ is 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, tetrahydro-2H-thiopyran,and morpholinyl, and the like) optionally substituted with one or more-Q₅-T₅.

For example, R₇ is 5 to 6-membered heterocycloalkyl optionallysubstituted with one or more -Q₅-T₅.

For example, R₇ is isopropyl.

For example, R₇ is pyrrolidinyl, piperidinyl, tetrahydropyran,tetrahydro-2H-thiopyranyl, cyclopentyl, or cyclohexyl, cycloheptyl, eachoptionally substituted with one or more -Q₅-T₅.

For example, R₇ is cyclopentyl cyclohexyl or tetrahydro-2H-thiopyranyl,each optionally substituted with one or more -Q₅-T₅.

For example, one or more -Q₅-T₅ are oxo.

For example, R₇ is 1-oxide-tetrahydro-2H-thiopyranyl or1,1-dioxide-tetrahydro-2H-thiopyranyl.

For example, Q is a bond and T₅ is amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino.

For example, Q5 is NHC(O) and T₅ is C₁-C₆ alkyl or C₁-C₆ alkoxy.

For example, T₄ is 4 to 7-membered heterocycloalkyl or C₃-C₈ cycloalkyland one or more -Q₅-T₅ are oxo.

For example, T₅ is H, halo, C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, or 4 to 7-membered heterocycloalkyl.

For example, Q is a bond and T₅ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or 4to 7-membered heterocycloalkyl.

For example, Q₅ is CO, S(O)₂, or NHC(O); and T₅ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, T₅ is C₁-C₆ alkyl or C₁-C₆ alkoxyl, each optionallysubstituted with halo, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, or C₃-C₈ cycloalkyl.

For example, Q5 is C₁-C₃ alkyl linker and T₅ is H or C₆-C₁₀ aryl.

For example, Q is C₁-C₃ alkyl linker and T₅ is C₃-C₈ cycloalkyl, 4 to7-membered heterocycloalkyl, or S(O)_(q)R_(q).

For example, R₁₁ is H.

For example, each of R₂ and R₄, independently, is H or C₁-C₆ alkyloptionally substituted with amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, or C₆-C₁₀ aryl.

For example, each of R₂ and R₄, independently is C₁-C₃ alkyl optionallysubstituted withC₁-C₆ alkoxyl.

For example, each of R₂ and R₄ is methyl.

For example, R₁ is H.

For example, R₁₂ is H, methyl, ethyl, ethenyl, or halo.

For example, R₁₂ is methyl.

For example, R₁₂ is ethyl.

For example, R₁₂ is ethenyl.

For example, R₈ is H, methyl, ethyl, or ethenyl.

For example, R₈ is methyl.

For example, R₈ is ethyl.

For example, R₈ is 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, tetrahydro-2H-thiopyran,morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like).

For example, R₈ is tetrahydropyran.

For example, R₈ is tetrahydropyran and R₇ is -Q₄-T₄, in which Q4 is abond or C₁-C₄ alkyl linker and T₄ is H, C₁-C₆ alkyl, C₃-C₈ cycloalkyl or4 to 7-membered heterocycloalkyl.

For example, Z₁ is NR₇R₈ or CR₇R₈R₁₄ wherein R₇ and R₈, together withthe atom to which they are attached, form a 4 to 11-memberedheterocycloalkyl ring having 1 to 3 heteroatoms (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, tetrahydro-2H-thiopyran,and morpholinyl, and the like) or C₃-C₈ cycloalkyl, each optionallysubstituted with one or more -Q₆-T₆.

For example, the ring formed by R₇ and R₈ is selected from the groupconsisting of azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl,piperazinyl, and cyclohexenyl, each optionally substituted with one-Q₆-T₆.

For example, -Q₆-T₆ is oxo.

For example, T₆ is H, halo, C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, or 4 to 7-membered heterocycloalkyl.

For example, Q6 is a bond and T₆ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or 4to 7-membered heterocycloalkyl.

For example, Q₆ is CO, S(O)₂, or NHC(O); and T₆ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, T₆ is C₁-C₆ alkyl or C₁-C₆ alkoxyl, each optionallysubstituted with halo, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, or C₃-C₈ cycloalkyl.

For example, Q₆ is C₁-C₃ alkyl linker and T₆ is H or C₆-C₁₀ aryl.

For example, Q₆ is C₁-C₃ alkyl linker and T₆ is C₃-C₈ cycloalkyl, 4 to7-membered heterocycloalkyl, or S(O)_(p)R_(p).

For example, each of R_(p) and R_(q), independently, is C₁-C₆ alkyl.

For example, R₁₃ is H or methyl.

For example, R₁₃ is H.

For example, R₃ is H.

For example, A⁻ is Br⁻.

For example, each of R₅, R₉, and R₁₀ is H.

Another subset of the compounds of Formula (II) includes those ofFormula (He):

The compounds of Formula (He) can include one or more of the followingfeatures:

For example, each of R_(a) and R_(b), independently is H or C₁-C₆ alkyloptionally substituted with one or more -Q₃-T₃.

For example, one of R_(a) and R_(b) is H.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms to the N atom (e.g., azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like) and the ring is optionally substituted with one or more-Q₃-T₃.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl,and the ring is optionally substituted with one or more -Q₃-T₃.

For example, one or more -Q₃-T₃ are oxo.

For example, Q₃ is a bond or unsubstituted or substituted C₁-C₃ alkyllinker.

For example, T₃ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃alkyl, OR_(d), COOR_(d), —S(O)₂R_(d), or —NR_(d)R_(c).

For example, one of R_(d) and R_(e) is H.

For example, R₇ is C₃-C₈ cycloalkyl or 4 to 7-membered heterocycloalkyl,each optionally substituted with one or more -Q₅-T₅.

For example, R₇ is piperidinyl, tetrahydropyran,tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl, orcycloheptyl, each optionally substituted with one or more -Q₅-T₅.

For example, R₇ is cyclopentyl cyclohexyl or tetrahydro-2H-thiopyranyl,each optionally substituted with one or more -Q₅-T₅.

For example, Q₅ is NHC(O) and T₅ is C₁-C₆ alkyl or C₁-C₆ alkoxy.

For example, one or more -Q₅-T₅ are oxo.

For example, R₇ is 1-oxide-tetrahydro-2H-thiopyranyl or1,1-dioxide-tetrahydro-2H-thiopyranyl.

For example, Q₅ is a bond and T₅ is amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino.

For example, Q₅ is CO, S(O)₂, or NHC(O); and T₅ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, R₈ is H, methyl, or ethyl.

The compounds suitable for use in the method of this invention alsoinclude compounds of Formula (III):

or a pharmaceutically acceptable salt or ester thereof. In Formula(III):

X₁′ is N or CR₁₁′;

X₂′ is N or CR₁₃′;

X₃ is N or C, and when X₃ is N, R₆′ is absent;

Z₂ is NR₇′R₈′, OR_(S)', S(O)_(a)′R₇′, or CR₇′R₈′R₁₄′, in which a′ is 0,1, or 2;

each of R₁′, R₅′, R₉′, and R₁₀′, independently, is H or C₁-C₆ alkyloptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5- or6-membered heteroaryl;

each of R₂′, R₃′, and R₄′, independently, is -Q₁′-T₁′, in which Q1′ is abond or C₁-C₃ alkyl linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₁′ is H, halo, hydroxyl, COOH, cyano,azido, or R_(S1)′, in which R_(S1)′ is C₁-C₃ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ alkoxyl, C(O)O—C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, 4 to12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, andR_(S1)′ is optionally substituted with one or more substituents selectedfrom the group consisting of halo, hydroxyl, oxo, COOH, C(O)O—C₁-C₆alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl;

R₆′ is H, halo, cyano, azido, OR_(a)′, —C(O)R_(a)′, —C(O)OR_(a)′,—C(O)NR_(a)′R_(b)′, —NR_(b)′C(O)R_(a)′, —S(O)_(b′)R_(a)′,—S(O)_(b′)NR_(a)′R_(b)′, or R_(S2)′, in which R_(S2)′ is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, or 4 to 12-memberedheterocycloalkyl, b′ is 0, 1, or 2, each of R_(a)′ and R_(b)′,independently is H or R_(S3)′, and R_(S3)′ is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl; or R_(a)′ and R_(b)′,together with the N atom to which they are attached, form a 4 to12-membered heterocycloalkyl ring having 0 or 1 additional heteroatom;and each of R_(S2)′, R_(S3)′, and the 4 to 12-membered heterocycloalkylring formed by R_(a)′ and R_(b)′, is optionally substituted with one ormore -Q₂′-T₂′, wherein Q₂′ is a bond or C₁-C₃ alkyl linker eachoptionally substituted with halo, cyano, hydroxyl or C₁-C₆ alkoxy, andT₂′ is H, halo, cyano, —OR_(c)′, —NR_(c)′R_(d)′, —C(O)R_(c)′,—C(O)OR_(c)′, —C(O)NR_(c)′R_(d)′, —NR_(d)′C(O)R_(c)′,—NR_(d)′C(O)OR_(c)′, —S(O)₂R_(c)′, —S(O)₂NR_(c)′R_(d)′, or R_(S4)′, inwhich each of R_(c)′ and R_(d)′, independently is H or R_(S5)′, each ofR_(S4)′ and R_(S5)′, independently, is C₁-C₆ alkyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, or R_(c)′ and R_(d)′, together with the N atom to which theyare attached, form a 4 to 12-membered heterocycloalkyl ring having 0 or1 additional heteroatom, and each of R_(S4)′, R_(S5)′, and the 4 to12-membered heterocycloalkyl ring formed by R_(c)′ and R_(d)′, isoptionally substituted with one or more -Q₃′-T₃′, wherein Q3′ is a bondor C₁-C₃ alkyl linker each optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₃′ is selected from the group consistingof halo, cyano, C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, OR_(e)′,COOR_(e)′, —S(O)₂R_(e)′, —NR_(e)′R_(f)′, and —C(O)NR_(e)′R_(f)′, each ofR_(e)′ and R_(f)′ independently being H or C₁-C₆ alkyl, or -Q₃′-T₃′ isoxo; or -Q₂′-T₂′ is oxo; provided that -Q₂′-T₂′ is not H;

R₇′ is -Q₄′-T₄′, in which Q₄′ is a bond, C₁-C₄ alkyl linker, or C₂-C₄alkenyl linker, each linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₄′ is H, halo, cyano, NR_(g)′R_(h)′,—OR_(g)′, —C(O)R_(g)′, —C(O)OR_(g)′, —C(O)NR_(g)′R_(h)′,—C(O)NR_(g)′OR_(h)′, —NR_(g)′C(O)R_(h)′, —S(O)₂R_(g)′, or R₅₆′, in whicheach of R_(g)′ and R_(h)′, independently is H or R_(S7)′, each of R₅₆′and R_(S7)′, independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5-or 6-membered heteroaryl, and each of R_(S6)′ and R_(S7)′ is optionallysubstituted with one or more -Q₅′-T₅′, wherein Q′ is a bond, C(O),C(O)NR_(k)′, NR_(k)′C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(k)′ being Hor C₁-C₆ alkyl, and T₅′ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(q′)R_(q)′ in which q′ is 0, 1, or 2 andR_(q)′ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₅′ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₅ is H,halo, hydroxyl, or cyano; or -Q₅′-T₅′ is oxo; provided that R₇′ is notH;

each of R₈′, R₁₁′, R₁₂′, and R₁₃′, independently, is H, halo, hydroxyl,COOH, cyano, R_(S8)′, OR_(S8)′, or COOR_(S8)′, in which R_(S8)′ is C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, 4 to 12-memberedheterocycloalkyl, amino, mono-C₁-C₆ alkylamino, or di-C₁-C₆ alkylamino,and R_(S8)′ is optionally substituted with one or more substituentsselected from the group consisting of halo, hydroxyl, COOH, C(O)O—C₁-C₆alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆alkylamino; or R₇′ and R₈′, together with the N atom to which they areattached, form a 4 to 12-membered heterocycloalkyl ring having 0 to 2additional heteroatoms, or R₇′ and R₈′, together with the C atom towhich they are attached, form C₃-C₈ cycloalkyl or a 4 to 12-memberedheterocycloalkyl ring having 1 to 3 heteroatoms, and each of the 4 to12-membered heterocycloalkyl rings or C₃-C₈ cycloalkyl formed by R₇′ andR₈′ is optionally substituted with one or more -Q₆′-T₆′, wherein Q6′ isa bond, C(O), C(O)NR_(m)′, NR_(m)′C(O), S(O)₂, or C₁-C₃ alkyl linker,R_(m)′ being H or C₁-C₆ alkyl, and T₆′ is H, halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, 5- or 6-membered heteroaryl, or S(O)_(p′)R_(p)′ inwhich p′ is 0, 1, or 2 and R_(p)′ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and T₆′ is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroarylexcept when T₆′ is H, halo, hydroxyl, or cyano; or -Q₆′-T₆′ is oxo; and

R₁₄′ is absent, H, or C₁-C₆ alkyl optionally substituted with one ormore substituents selected from the group consisting of halo, hydroxyl,COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl.

One subset of the compounds of Formula (III) includes those of Formula(Ma):

Another subset of the compounds of Formula (III) includes those ofFormula (IIIb), (IIIc), or (IIId):

The compounds of Formulae (III), (IIIa), (IIIb), (IIIc), and (IIId) caninclude one or more of the following features:

For example, the compounds of Formula (III) are not

-   N-(5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-2-methylphenyl)furan-2-carboxamide,-   N,N′-(5-(((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamoyl)-1,3-phenylene)diacetamide,-   N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-pivalamidobenzamide,-   3-(3,4-dihydro-2H-benzo[b][1,4]dioxepine-7-sulfonamido)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzamide,-   N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,5-dimethoxybenzamide,-   N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4,5-trimethoxybenzamide,-   3-allyl-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-4,5-dimethoxybenzamide,-   4-(2-amino-2-oxoethoxy)-3-chloro-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methoxybenzamide,-   3-chloro-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-4-hydroxy-5-methoxybenzamide,    or-   3-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methoxy-4-propoxybenzamide.

For example, X₁′ is CR₁₁′ and X₂′ is CR₁₃′.

For example, X₁′ is CR₁₁′ and X₂′ is N.

For example, X₁′ is N and X₂′ is CR₁₃′.

For example, X₁′ is N and X₂′ is N.

For example, X₃ is C.

For example, X₃ is N and R₆′ is absent.

For example, Z₂ is NR₇′R₈′.

For example, Z₂ is CR₇′R₈′R₁₄′.

For example, Z₂ is OR₇′.

For example, Z₂ is S(O)_(a)′R₇′, in which a′ is 0, 1, or 2.

For example, R₆′ is H.

For example, R₆′ is halo (e.g., fluorine, chlorine, bromine, andiodine).

For example, R₆′ is C₁-C₃ alkyl optionally substituted with one or more-Q₂′-T₂′.

For example, R₆′ is CF₃.

For example, R₆′ is C₂-C₆ alkenyl, C₂-C₆ alkynyl, or C₃-C₆ cycloalkyleach optionally substituted with one or more -Q₂′-T₂′.

For example, R₆′ is ethenyl.

For example, R₆′ is ethynyl.

For example, R₆′ is ethynyl substituted with one or more -Q₂′-T₂′, inwhich Q₂′ is a bond or C₁-C₃ alkyl linker and T₂′ is C₁-C₆ alkyl, C₃-C₆cycloalkyl, or 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like) optionally substituted with one or more-Q₃′-T₃′.

For example, R₆′ is cyano.

For example, R₆′ is azido.

For example, R₆′ is C(O)H.

For example, R₆′ is OR_(a)′ or —C(O)R_(a)′.

For example, R_(a)′ is C₃-C₆ alkyl or 4 to 7-membered heterocycloalkyl(e.g., azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl,tetrahyrofuranyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like), which is optionally substituted with one ormore -Q₂′-T₂′.

For example, R₆′ is 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like) optionally substituted with one or more-Q₂′-T₂′.

For example, R₆′ is piperidinyl, 2,2,6,6-tetramethyl-piperidinyl,1,2,3,6-tetrahydropyridinyl,2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl, piperazinyl,morpholinyl, tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, orpyrrolidinyl, each of which is optionally substituted with one or more-Q₂′-T₂′.

For example, R₆′ is 4 to 7-membered heterocycloalkyl optionallysubstituted with one or more -Q₂′-T₂′, and -Q₂′-T₂′ is oxo or Q2′ is abond and T₂′ is —OR_(c)′, —NR_(c)′R_(d)′, —C(O)R_(c)′, —C(O)OR_(c)′,—S(O)₂R_(c)′, C₁-C₆ alkyl, or 4 to 7-membered heterocycloalkyl, each ofwhich is optionally substituted with one or more -Q₃′-T₃′ when R_(c)′ orR_(d)′ is not H.

For example, R₆′ is —C(O)R_(a)′, —C(O)OR_(a)′, —C(O)NR_(a)′R_(b)′,—NR_(b)′C(O)R_(a)′, —SR_(a)′, —S(O)₂R_(a)′, or —S(O)₂NR_(a)′R_(b)′.

For example, each of R_(a)′ and R_(b)′, independently is H, C₁-C₆ alkyl,or C₃-C₈ cycloalkyl optionally substituted with one or more -Q₂′-T₂′.

For example, one of R_(a)′ and R_(b)′ is H.

For example, R_(a)′ and R_(b)′, together with the N atom to which theyare attached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms to the N atom (e.g., azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,2,5-diazabicyclo[2.2.1]heptanyl, and morpholinyl, and the like) and thering is optionally substituted with one or more -Q₂′-T₂′.

For example, -Q₂′-T₂′ is oxo.

For example, Q2′ is a bond.

For example, Q2′ is an unsubstituted C₁-C₃ alkyl linker.

For example, T₂′ is C₁-C₆ alkyl or C₆-C₁₀ aryl, each optionallysubstituted with one or more -Q₃′-T_(3′.)

For example, T₂′ is an unsubstituted substituted straight chain C₁-C₆ orbranched C₃-C₆ alkyl, including but not limited to, methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl andn-hexyl.

For example, T₂′ is phenyl.

For example, T₂′ is halo (e.g., fluorine, chlorine, bromine, andiodine).

For example, T₂′ is 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like) optionally substituted with one or more-Q₃′-T₃′.

For example, T₂′ is —OR_(c)′, —C(O)R_(c)′, —C(O)OR_(c)′, or—S(O)₂R_(c)′.

For example, is C₁-C₆ alkyl or 4 to 7-membered heterocycloalkyl (e.g.,azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl,tetrahyrofuranyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like), which is optionally substituted with one ormore -Q₃′-T₃′.

For example, each of R_(c)′ and R_(d)′, independently is H or C₁-C₆alkyl optionally substituted with one or more -Q₃′-T₃′.

For example, R_(c)′ is H.

For example, R_(d)′ is H.

For example, R_(c)′ and R_(d)′, together with the N atom to which theyare attached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms to the N atom (e.g., azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,2,5-diazabicyclo[2.2.1]heptanyl, and morpholinyl, and the like) and thering is optionally substituted with one or more -Q₃′-T_(3′.)

For example, Q₂′ is a bond and T₂′ is —OR_(c)′, —NR_(c)′R_(d)′,—C(O)R_(c)′, —C(O)OR_(c)′, —S(O)₂R_(c)′, C₁-C₆ alkyl, or 4 to 7-memberedheterocycloalkyl, each of which is optionally substituted with one ormore -Q₃′-T₃′ when R_(c)′ or R_(d)′ is not H.

For example, -Q₃′-T₃′ is oxo.

For example, T₂′ is 4 to 7-membered heterocycloalkyl or C₃-C₈ cycloalkyland one or more -Q₃′-T₃′ are oxo.

For example, Q3′ is a bond or unsubstituted or substituted C₁-C₃ alkyllinker.

For example, T₃′ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃alkyl, OR_(e)′, COOR_(e)′, —S(O)₂R_(e)′, —NR_(e)′R_(f)′, or—C(O)NR_(e)′R_(f)′.

For example, one of R_(d)′ and R_(e)′ is H.

For example, Q₃′ is a bond or C₁-C₃ alkyl linker and T₃′ is selectedfrom the group consisting of C₁-C₃ alkyl, halo, OR_(e)′, —S(O)₂R_(e)′,—NR_(e)′R_(f)′, and —C(O)NR_(c)′R_(f)′.

For example, Q₃′ is a bond or C₁-C₃ alkyl linker and T₃′ is selectedfrom the group consisting of C₁-C₃ alkyl, OR_(e)′, —S(O)₂R_(e)′, or—NR_(e)′R_(f)′.

For example, R_(e)′ is H.

For example, R_(f)′ is H.

For example, R₇′ is —C(O)R_(g)′.

For example, R₇′ is —C(O)R_(g)′, in which R_(g)′ is C₃-C₈ cycloalkyl, 4to 7-membered heterocycloalkyl, C₃-C₈ cycloalkyl.

For example, R₇′ is C₆-C₁₀ aryl substituted with one or more -Q₅′-T₅′.

For example, R₇′ is phenyl optionally substituted with one or more-Q₅′-T₅′.

For example, R₇′ is C₁-C₆ alkyl optionally substituted with one or more-Q₅′-T₅′.

For example, R₇′ is C₃-C₈ cycloalkyl optionally substituted with one ormore -Q₅′-T₅′.

For example, R₇′ is 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like) optionally substituted with one or more-Q₅′-T₅′.

For example, R₇′ is 5 to 6-membered heterocycloalkyl optionallysubstituted with one or more -Q₅′-T₅′.

For example, R₇′ is isopropyl.

For example, R₇′ is pyrrolidinyl, piperidinyl, tetrahydropyran,cyclopentyl, or cyclohexyl, cycloheptyl, each optionally substitutedwith one -Q₅′-T₅′.

For example, R₇′ is cyclopentyl or cyclohexyl, each optionallysubstituted with one -Q′-T₅′.

For example, Q₅′ is NHC(O) and T₅′ is C₁-C₆ alkyl or C₁-C₆ alkoxy.

For example, -Q₅′-T₅′ is oxo.

For example, T₄′ is 4 to 7-membered heterocycloalkyl, C₃-C₈ cycloalkyl,or C₆-C₁₀ aryl, and one or more -Q₅′-T₅′ are oxo.

For example, R₇′ is 1-oxide-tetrahydro-2H-thiopyranyl or1,1-dioxide-tetrahydro-2H-thiopyranyl.

For example, R₇′ is cyclohexanonyl, e.g., cyclohexanon-4-yl.

For example, T₅′ is H, halo, C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, or 4 to 7-membered heterocycloalkyl.

For example, Q5′ is a bond and T₅′ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or4 to 7-membered heterocycloalkyl.

For example, Q₅′ is a bond and T₅′ is 5- or 6-membered heteroaryl,amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, T₅′ being optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, C₁-C₆ alkoxyl, or C₃-C₈ cycloalkyl.

For example, Q₅′ is CO, S(O)₂, or NHC(O); and T₅′ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, T₅′ is C₁-C₆ alkyl or C₁-C₆ alkoxyl, each optionallysubstituted with halo, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, or C₃-C₈ cycloalkyl.

For example, Q₅′ is C₁-C₃ alkyl linker and T₅′ is H or C₆-C₁₀ aryl.

For example, Q₅′ is C₁-C₃ alkyl linker and T₅′ is C₃-C₈ cycloalkyl, 4 to7-membered heterocycloalkyl, or S(O)_(q)′R_(q)′.

For example, R₆′ is halo (e.g., fluorine, chlorine, bromine, and iodine)and Z₂ is S(O)_(a)′R₇′, in which a′ is 0, 1, or 2 and R₇′ is C₁-C₆ alkyl(e.g., methyl, ethyl, n-propyl, i-propyl, butyl, or t-butyl), C₃-C₈cycloalkyl (e.g., cyclopentyl, cyclohexyl, or cycloheptyl) or 4 to7-membered heterocycloalkyl (e.g., azetidinyl, oxetanyl, thietanyl,pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl,isoxazolidinyl, triazolidinyl, tetrahyrofuranyl, piperidinyl,1,2,3,6-tetrahydropyridinyl, piperazinyl, tetrahydro-2H-pyranyl,3,6-dihydro-2H-pyranyl, tetrahydro-2H-thiopyranyl, 1,4-diazepanyl,1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,2,5-diazabicyclo[2.2.1]heptanyl, and morpholinyl, and the like) and R₇′is optionally substituted with one or more -Q₅′-T₅′.

For example, R₆′ is halo (e.g., fluorine, chlorine, bromine, and iodine)and Z₂ is OR₇′ in which R₇′ is 4 to 7-membered heterocycloalkyl (e.g.,azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl,tetrahyrofuranyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like) and R₇′ is optionally substituted with one ormore -Q₅′-T₅′.

For example, R₁₁′ is H.

For example, each of R₂′ and R₄′, independently, is H or C₁-C₆ alkyloptionally substituted with azido, halo, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, or C₆-C₁₀ aryl.

For example, each of R₂′ and R₄′, independently is C₁-C₃ alkyloptionally substituted with C₁-C₆ alkoxyl.

For example, each of R₂′ and R₄′ is methyl.

For example, R₁′ is H.

For example, R₁′ is C₁-C₆ alkyl optionally substituted with azido, halo,amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, or C₆-C₁₀ aryl.

For example, R₁₂′ is H, methyl, ethyl, ethenyl, or halo.

For example, R₁₂′ is methyl.

For example, R₁₂′ is ethyl.

For example, R₁₂′ is ethenyl or propenyl.

For example, R₁₂′ is methoxyl.

For example, R₈′ is H, methyl, ethyl, or ethenyl.

For example, R₈′ is methyl.

For example, R₈′ is ethyl.

For example, R₈′ is propyl.

For example, R₈′ is ethenyl or propenyl.

For example, R₈′ is C₁-C₆ alkyl substituted with one or moresubstituents selected from the group consisting of halo (e.g., F, Cl, orBr), hydroxyl, or C₁-C₆ alkoxyl.

For example, R₈′ is 4 to 7-membered optionally substitutedheterocycloalkyl (e.g., azetidinyl, oxetanyl, thietanyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, tetrahyrofuranyl, piperidinyl,1,2,3,6-tetrahydropyridinyl, piperazinyl, tetrahydro-2H-pyranyl,3,6-dihydro-2H-pyranyl, tetrahydro-2H-thiopyranyl, 1,4-diazepanyl,1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl,2,5-diazabicyclo[2.2.1]heptanyl, and morpholinyl, and the like).

For example, R₈′ is piperidinyl.

For example, R₈′ is 4 to 7-membered optionally substitutedheterocycloalkyl and R₇′ is -Q₄′-T₄′, in which Q₄′ is a bond or C₁-C₄alkyl linker and T₄′ is H, C₁-C₆ alkyl, C₃-C₈ cycloalkyl or 4 to7-membered heterocycloalkyl.

For example, Z₂ is NR₇′R₈′ or CR₇′R₈′R₁₄′ wherein R₇′ and R₈′, togetherwith the atom to which they are attached, form a 4 to 11-memberedheterocycloalkyl ring having 1 to 3 heteroatoms (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,morpholinyl, and the like) or C₃-C₈ cycloalkyl, each optionallysubstituted with one or more -Q₆′-T₆′.

For example, the ring formed by R₇′ and R₈′ is selected from the groupconsisting of azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl,piperazinyl, and cyclohexenyl, each optionally substituted with one-Q₆′-T₆′.

For example, -Q₆′-T₆′ is oxo.

For example, T₆′ is H, halo, C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, or 4 to 7-membered heterocycloalkyl.

For example, Q₆′ is a bond and T₆ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or 4to 7-membered heterocycloalkyl.

For example, Q₆′ is CO, S(O)₂, or NHC(O); and T₆′ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, T₆′ is C₁-C₆ alkyl or C₁-C₆ alkoxyl, each optionallysubstituted with halo, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, or C₃-C₈ cycloalkyl.

For example, Q6′ is C₁-C₃ alkyl linker and T₆′ is H or C₆-C₁₀ aryl.

For example, Q6′ is C₁-C₃ alkyl linker and T₆′ is C₃-C₈ cycloalkyl, 4 to7-membered heterocycloalkyl, or S(O)_(p′)R_(p)′.

For example, each of R_(p)′ and R_(q)′, independently, is C₁-C₆ alkyl.

For example, R₆′ is —S(O)_(b)′R_(a)′ or azido, in which b′ is 0, 1, or 2and R_(a)′ is C₁-C₆ alkyl or C₃-C₈ cycloalkyl; and Z₂ is NR₇′R₈′, inwhich R₇′ is C₃-C₈ cycloalkyl (e.g., cyclopentyl, cyclohexyl, orcycloheptyl) or 4 to 7-membered heterocycloalkyl (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andmorpholinyl, and the like), each optionally substituted with one or more-Q₅′-T₅′ and R₈′ is H or C₁-C₆ alkyl (e.g., methyl, ethyl, n-propyl,i-propyl, butyl, or t-butyl).

For example, R₆′ is halo (e.g., fluorine, chlorine, bromine, and iodine)and Z₂ is NR₇′R₈′ or CR₇′R₈′R₁₄′ wherein R₇′ and R₈′, together with theatom to which they are attached, form a 4 to 11-memberedheterocycloalkyl ring having 1 to 3 heteroatoms (e.g., azetidinyl,oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl,tetrahydro-2H-thiopyranyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,morpholinyl, and the like) or C₃-C₈ cycloalkyl, each optionallysubstituted with one or more -Q₆′-T₆′.

For example, R₁₃′ is H or methyl.

For example, R₁₃′ is H.

For example, R₃′ is H.

For example, each of R₅′, R₉′, and R₁₀′ is H.

Other compounds suitable for the methods of the invention are describedin PCT/US2012/026953, filed on Feb. 28, 2012; U.S. ProvisionalApplication Ser. Nos. 61/474,821, filed on Apr. 13, 2011; 61/474,825,filed on Apr. 13, 2011; 61/499,595, filed on Jun. 21, 2011; and61/505,676, filed on Jul. 8, 2011, the contents of which are herebyincorporated by reference in their entireties.

Exemplary EZH2 inhibitor compounds of the present invention are shown inTable 1. In the table below, each occurrence of

should be construed as

TABLE 1 Com- pound Number Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

Unless otherwise indicated, the term “substituted” means substituted byone or more defined groups. In the case where groups may be selectedfrom a number of alternative groups the selected groups may be the sameor different.

The term “independently” means that where more than one substituent isselected from a number of possible substituents, those substituents maybe the same or different.

An “effective amount” means that amount of a drug or pharmaceuticalagent that will elicit the biological or medical response of a tissue,system, animal or human that is being sought, for instance, by aresearcher or clinician. Furthermore, the term “therapeuticallyeffective amount” means any amount which, as compared to a correspondingsubject who has not received such amount, results in improved treatment,healing, prevention, or amelioration of a disease, disorder, or sideeffect, or a decrease in the rate of advancement of a disease ordisorder. The term also includes within its scope amounts effective toenhance normal physiological function.

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C_(I), C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups.Examples of alkyl include, moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in another embodiment, a straight chain or branched alkylhas four or fewer carbon atoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g., fused,bridged; or spiro rings) system having 3 to 30 carbon atoms (e.g.,C₃-C₁₀). Examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl.The term “heterocycloalkyl” refers to a saturated or unsaturatednonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic (fused,bridged, or spiro rings), or 11-14 membered tricyclic ring system(fused, bridged, or spiro rings) having one or more heteroatoms (such asO, N, S, or Se), unless specified otherwise. Examples ofheterocycloalkyl groups include, but are not limited to, piperidinyl,piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl,indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, tetrahyrofuranyl, oxiranyl, azetidinyl, oxetanyl,thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl,dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated divalent aliphatic hydrocarbongroups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbongroups. For example, C₁-C₆ alkyl linker is intended to include C₁, C₂,C₃, C₄, C₅ and C₆ alkyl linker groups. Examples of alkyl linker include,moieties having from one to six carbon atoms, such as, but not limitedto, methyl (—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenylgroups. In certain embodiments, a straight chain or branched alkenylgroup has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ forstraight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkenyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. Incertain embodiments, a straight chain or branched alkynyl group has sixor fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents. For example, substituted heterocycloalkylincludes those substituted with one or more alkyl groups, such as2,2,6,6-tetramethyl-piperidinyl and2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5-, 6-, or7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g. 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizirie.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can besubstituted at one or more ring positions (e.g., the ring-forming carbonor heteroatom such as N) with such substituents as described above, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroarylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. Carbocycle includes cycloalkyl and aryl. For example, aC₃-C₁₄ carbocycle is intended to include a monocyclic, bicyclic ortricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbonatoms. Examples of carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are alsoincluded in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and Spiro ringsare also included.

As used herein, “heterocycle” or “heterocyclic group” includes any ringstructure (saturated, unsaturated, or aromatic) which contains at leastone ring heteroatom (e.g., N, O or S). Heterocycle includesheterocycloalkyl and heteroaryl. Examples of heterocycles include, butare not limited to, morpholine, pyrrolidine, tetrahydrothiophene,piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine, andtetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is pxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R_(I) at each occurrence is selected independently from thedefinition of R₁. Also, combinations of substituents and/or variablesare permissible, but only if such combinations result in stablecompounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to unsubstituted orsubstituted —NH₂. “Alkylamino” includes groups of compounds whereinnitrogen of —NH₂ is bound to at least one alkyl group. Examples ofalkylamino groups include benzylamino, methylamino, ethylamino,phenethylamino, etc. “Dialkylamino” includes groups wherein the nitrogenof —NH₂ is bound to at least two additional alkyl groups. Examples ofdialkylamino groups include, but are not limited to, dimethylamino anddiethylamino. “Arylamino” and “diarylamino” include groups wherein thenitrogen is bound to at least one or two aryl groups, respectively.“Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxy substitutedwith amino. “Alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl”refers to an amino group which is bound to at least one alkyl group andat least one aryl group. “Alkaminoalkyl” refers to an alkyl, alkenyl, oralkynyl group bound to a nitrogen atom which is also bound to an alkylgroup. “Acylamino” includes groups wherein nitrogen is bound to an acylgroup. Examples of acylamino include, but are not limited to,alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”;“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

The term “optionally” means that the subsequently described event(s) mayor may not occur, and includes both event(s), which occur, and eventsthat do not occur.

Herein, the term “pharmaceutically-acceptable salts” refers to saltsthat retain the desired biological activity of the subject compound andexhibit minimal undesired toxicological effects. Thesepharmaceutically-acceptable salts may be prepared in situ during thefinal isolation and purification of the compound, or by separatelyreacting the purified compound in its free acid or free base form with asuitable base or acid, respectively.

By the term “co-administering” and derivatives thereof as used herein ismeant either simultaneous administration or any manner of separatesequential administration of one or more additional pharmaceuticallyactive compounds, whether for treating cancer, the side effects ofcancer or cancer therapy, or some other disease. Preferably, if theadministration is not simultaneous, the compounds are administered in aclose time proximity to each other. Furthermore, it does not matter ifthe compounds are administered in the same dosage form, e.g. onecompound may be administered topically and another compound may beadministered orally.

The compounds of Formulae (I)-(III) include the compounds themselves, aswell as their salts, their solvates, their N-oxides, and their prodrugs,if applicable. In certain embodiments, compounds according to Formulae(I)-(III) may contain an acidic functional group, one acidic enough toform salts. Representative salts include pharmaceutically acceptablemetal salts such as sodium, potassium, lithium, calcium, magnesium,aluminum, and zinc salts; carbonates and bicarbonates of apharmaceutically-acceptable metal salts such as sodium, potassium,lithium, calcium, magnesium, aluminum, and zinc;pharmaceutically-acceptable organic primary, secondary, and tertiaryamines including aliphatic amines, aromatic amines, aliphatic diamines,and hydroxy alkylamines such as methylamine, ethylamine,2-hydroxyethylamine, diethylamine, triethylamine, ethylenediamine,ethanolamine, diethanolamine, and cyclohexylamine.

In certain embodiments, compounds according to Formulae (I)-(III) maycontain a basic functional group and are therefore capable of formingpharmaceutically-acceptable acid addition salts by treatment with asuitable acid. Suitable acids include pharmaceutically-acceptableinorganic acids and pharmaceutically-acceptable organic acids.Representative pharmaceutically acceptable acid addition salts includehydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate,sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate,propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate,acrylate, fumarate, malate, tartrate, citrate, salicylate,p-aminosalicylate, glycollate, lactate, heptanoate, phthalate, oxalate,succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate,formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate,malonate, laurate, glutarate, glutamate, estolate, methanesulfonate(mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate,benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate(tosylate) and napthalene-2-sulfonate.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present invention maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present invention, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of keto-enol equilibria is betweenpyridin-2(1H)-ones and the corresponding pyridin-2-ols, as shown below.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms, includingmixtures thereof, are intended to be included in the scope of thepresent invention, and the naming of the compounds does not exclude anytautomer form.

The compounds of Formulae (I)-(III) may be prepared in crystalline ornon-crystalline form, and, if crystalline, may optionally be solvated,e.g., as the hydrate. This invention includes within its scopestoichiometric solvates (e.g., hydrates) as well as compounds containingvariable amounts of solvent (e.g., water).

Certain of the compounds described herein may contain one or more chiralatoms, or may otherwise be capable of existing as two enantiomers. Thecompounds claimed below include mixtures of enantiomers as well aspurified enantiomers or enantiomerically enriched mixtures. Alsoincluded within the scope of the invention are the individual isomers ofthe compounds represented by Formulae (I)-(III), or claimed below, aswell as any wholly or partially equilibrated mixtures thereof. Thepresent invention also covers the individual isomers of the claimedcompounds as mixtures with isomers thereof in which one or more chiralcenters are inverted.

Where there are different isomeric forms they may be separated orresolved one from the other by conventional methods, or any given isomermay be obtained by conventional synthetic methods or by stereospecificor asymmetric syntheses.

While it is possible that, for use in therapy, a compound of Formulae(I)-(III), as well as salts, solvates, N-oxides, and the like, may beadministered as a neat preparation, i.e., no additional carrier, themore usual practice is to present the active ingredient confected with acarrier or diluent. Accordingly, the invention further providespharmaceutical compositions, which includes a compound of Formulae(I)-(III) and salts, solvates and the like, and one or morepharmaceutically acceptable carriers, diluents, or excipients. Thecompounds of Formulae (I)-(III) and salts, solvates, N-oxides, etc, areas described above. The carrier(s), diluent, or excipient(s) must beacceptable in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Inaccordance with another aspect of the invention there is also provided aprocess for the preparation of a pharmaceutical formulation includingadmixing a compound of the Formulae (I)-(III), or salts, solvates,N-oxides, etc, with one or more pharmaceutically acceptable carriers,diluents or excipients.

It will be appreciated by those skilled in the art that certainprotected derivatives of compounds of Formulae (I)-(III), which may bemade prior to a final deprotection stage, may not possesspharmacological activity as such, but may, in certain instances, beadministered orally or parenterally and thereafter metabolized in thebody to form compounds of the invention which are pharmacologicallyactive. Such derivatives may therefore be described as “prodrugs”.Further, certain compounds of the invention may act as prodrugs of othercompounds of the invention. All protected derivatives and prodrugs ofcompounds of the invention are included within the scope of theinvention. It will further be appreciated by those skilled in the art,that certain moieties, known to those skilled in the art as“pro-moieties” may be placed on appropriate functionalities when suchfunctionalities are present within compounds of the invention. Preferredprodrugs for compounds of the invention include: esters, carbonateesters, hemi-esters, phosphate esters, nitro esters, sulfate esters,sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides,ethers, acetals, and ketals.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) are aryl- or heteroaryl-substituted benzene compounds, andhave Formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides; tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

Such compounds also include Compound 75

and pharmaceutically acceptable salts thereof.

The invention further includes a pharmaceutical composition comprisingCompound 75

or a pharmaceutically acceptable salt thereof.

An EZH2 antagonist and optionally other therapeutics can be administeredper se (neat) or in the form of a pharmaceutically acceptable salt. Whenused in medicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts can conveniently be used toprepare pharmaceutically acceptable salts thereof.

Compounds useful in accordance with the invention may be provided assalts with pharmaceutically compatible counterions (i.e.,pharmaceutically acceptable salts). A “pharmaceutically acceptable salt”means any non-toxic salt that, upon administration to a recipient, iscapable of providing, either directly or indirectly, a compound or aprodrug of a compound useful in accordance with this invention. A“pharmaceutically acceptable counterion” is an ionic portion of a saltthat is not toxic when released from the salt upon administration to asubject. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, and succinic acids. Salts tend to be moresoluble in water or other protic solvents than their corresponding freebase forms. The present invention includes the use of such salts.

Pharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid and hydrobromic acid, andalso those formed with organic acids such as maleic acid. For example,acids commonly employed to form pharmaceutically acceptable saltsinclude inorganic acids such as hydrogen bisulfide, hydrochloric,hydrobromic, hydroiodic, sulfuric and phosphoric acid, as well asorganic acids such as para-toluenesulfonic, salicylic, tartaric,bitartaric, ascorbic, maleic, besylic, fumaric, gluconic, glucuronic,formic, glutamic, methanesulfonic, ethanesulfonic, benzenesulfonic,lactic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric,benzoic and acetic acid, and related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,terephathalate, sulfonate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate,glycolate, maleate, tartrate, methanesulfonate, propanesulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and thelike.

Suitable bases for forming pharmaceutically acceptable salts with acidicfunctional groups include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkylamines;dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), such as mono-, bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-dialkyl-N-(hydroxy alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

Certain compounds useful in accordance with the invention and theirsalts may exist in more than one crystalline form (i.e., polymorph); thepresent invention includes the use of each of the crystal forms andmixtures thereof.

Certain compounds useful in accordance with the invention may containone or more chiral centers, and exist in different optically activeforms. When compounds useful in accordance with the invention containone chiral center, the compounds exist in two enantiomeric forms and thepresent invention includes the use of both enantiomers and mixtures ofenantiomers, such as racemic mixtures thereof. The enantiomers may beresolved by methods known to those skilled in the art; for example,enantiomers may be resolved by formation of diastereoisomeric saltswhich may be separated, for example, by crystallization; formation ofdiastereoisomeric derivatives or complexes which may be separated, forexample, by crystallization, gas-liquid or liquid chromatography;selective reaction of one enantiomer with an enantiomer-specificreagent, for example, via enzymatic esterification; or gas-liquid orliquid chromatography in a chiral environment, for example, on a chiralsupport (e.g., silica with a bound chiral ligand) or in the presence ofa chiral solvent. Where the desired enantiomer is converted into anotherchemical entity by one of the separation procedures described above, afurther step may be used to liberate the desired purified enantiomer.Alternatively, specific enantiomers may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer into the other by asymmetrictransformation.

When a compound useful in accordance with the invention contains morethan one chiral center, it may exist in diastereoisomeric forms. Thediastereoisomeric compounds may be separated by methods known to thoseskilled in the art (for example, chromatography or crystallization) andthe individual enantiomers may be separated as described above. Thepresent invention includes the use of various diastereoisomers ofcompounds useful in accordance with the invention, and mixtures thereof.Compounds useful in accordance with the invention may exist in differenttautomeric forms or as different geometric isomers, and the presentinvention includes the use of each tautomer and/or geometric isomer ofcompounds useful in accordance with the invention, and mixtures thereof.Compounds useful in accordance with the invention may exist inzwitterionic form. The present invention includes the use of eachzwitterionic form of compounds useful in accordance with the invention,and mixtures thereof.

Kits

An EZH2 antagonist may, if desired, be presented in a kit (e.g., a packor dispenser device) which may contain one or more unit dosage formscontaining the EZH2 antagonist. The pack may for example comprise metalor plastic foil, such as a blister pack. The pack or dispenser devicemay be accompanied by instructions for administration. Compositionscomprising an EZH2 antagonist of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Instructions for use may also be provided.

Also provided herein are kits comprising a plurality of methylationdetection reagents that detect the methylated H3-K27. For example, thekit includes mono-methylated H3-K27, di-methylated H3-K27 andtri-methylated H3-K27 detection reagents. The detection reagent is forexample antibodies or fragments thereof, polypeptide or aptamers.

A kit may also include an EZH2 mutant detection reagent, e.g., nucleicacids that specifically identify a mutant EZH2 nucleic acid sequence byhaving homologous nucleic acid sequences, such as oligonucleotidesequences, complementary to a portion of the mutant EZH2 nucleic acidsequence or antibodies to proteins encoded by the mutant EZH2 nucleicacids packaged together in the form of a kit. The oligonucleotides canbe fragments of the EZH2 gene. For example the oligonucleotides can be200, 150, 100, 50, 25, 10 or less nucleotides in length. The kit maycontain in separate containers an aptamer or an antibody, controlformulations (positive and/or negative), and/or a detectable label suchas fluorescein, green fluorescent protein, rhodamine, cyanine dyes,Alexa dyes, luciferase, radiolabels, among others. Instructions (e.g.,written, tape, VCR, CD-ROM, etc.) for carrying out the assay may beincluded in the kit. The assay may for example be in the form of aWestern Blot analysis, Immunohistochemistry (IHC), immunofluorescence(IF), sequencing and Mass spectrometry (MS) as known in the art.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. All definitions, as defined andused herein, supersede dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The terms “co-administration” and “co-administering” refer to bothconcurrent administration (administration of two or more therapeuticagents at the same time) and time varied administration (administrationof one or more therapeutic agents at a time different from that of theadministration of an additional therapeutic agent or agents), as long asthe therapeutic agents are present in the patient to some extent at thesame time.

The term “precancerous condition” or “premalignant condition” refers toa disease, syndrome, or finding that, if left untreated, may lead tocancer. It is a generalized state associated with a significantlyincreased risk of cancer.

The term “treating” as used herein refers to alleviate of at least onesymptom of the disease, disorder or condition. The term encompasses theadministration and/or application of one or more compounds describedherein, to a subject, for the purpose of providing management of, orremedy for a condition. “Treatment” for the purposes of this disclosure,may, but does not have to, provide a cure; rather, “treatment” may be inthe form of management of the condition. When the compounds describedherein are used to treat unwanted proliferating cells, includingcancers, “treatment” includes partial or total destruction of theundesirable proliferating cells with minimal destructive effects onnormal cells. A desired mechanism of treatment of unwanted rapidlyproliferating cells, including cancer cells, at the cellular level isapoptosis.

The term “preventing” as used herein includes either preventing orslowing the onset of a clinically evident disease progression altogetheror preventing or slowing the onset of a preclinically evident stage of adisease in individuals at risk. This includes prophylactic treatment ofthose at risk of developing a disease.

The term “subject” as used herein for purposes of treatment includes anyhuman subject who has been diagnosed with, has symptoms of, or is atrisk of developing a cancer or a precancerous condition. For methods ofprevention the subject is any human subject. To illustrate, for purposesof prevention, a subject may be a human subject who is at risk of or isgenetically predisposed to obtaining a disorder characterized byunwanted, rapid cell proliferation, such as cancer. The subject may beat risk due to exposure to carcinogenic agents, being geneticallypredisposed to disorders characterized by unwanted, rapid cellproliferation, and so on.

Except as otherwise indicated, standard methods can be used for theproduction of recombinant and synthetic polypeptides, fusion proteins,antibodies or antigen-binding fragments thereof, manipulation of nucleicacid sequences, production of transformed cells, and the like. Suchtechniques are known to those skilled in the art. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Cold SpringHarbor, N.Y., 2001); F. M. Ausubel et al. Current Protocols in MolecularBiology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc.,New York).

The term “EZH2 polypeptide” encompasses functional fragments of thefull-length polypeptides and functional equivalents of either of theforegoing that have substantially similar or substantially identicalamino acid sequences (at least about 75%, 80%, 85%, 90%, 95% 98% or moreamino acid sequence similarity or identity), where the functionalfragment or functional equivalent retains one or more of the functionalproperties of the native polypeptide.

By “functional” it is meant that the polypeptide (or nucleic acid) hasthe same or substantially similar activity with respect to one or moreof the biological properties of the native polypeptide (or nucleicacid), e.g., at least about 50%, 75%, 85%, 90%, 95% or 98% or more ofthe activity of the native polypeptide (or nucleic acid).

The term “modulate” (and grammatical equivalents) refers to an increaseor decrease in activity. In particular embodiments, the term “increase”or “enhance” (and grammatical equivalents) means an elevation by atleast about 25%, 50%, 75%, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold or more. In particular embodiments, the terms “decrease” or“reduce” (and grammatical equivalents) means a diminishment by at leastabout 25%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or more. In someembodiments, the indicated activity, substance or other parameter is notdetectable. Specifically provided are antagonists of EZH2.

The term “pharmacodynamic marker” refers to a molecular marker of drugresponse that can be measured in patients receiving the drug. The markershould be a direct measure of modulation of the drug target and be ableto show quantitative changes in response to dose. A potentialpharmacodynamic marker for EZH2 antagonists could be levels of histoneH3-K27 methylation in disease or surrogate tissue.

As used herein, the term “responsiveness” is interchangeable with terms“responsive”, “sensitive”, and “sensitivity”, and it is meant that asubject showing therapeutic response when administered an EZH inhibitor,e.g., tumor cells or tumor tissues of the subject undergo apoptosisand/or necrosis, and/or display reduced growing, dividing, orproliferation.

The term “control” or “reference” refers to methylation levels (e.g.,monomethylation level, dimethylation level or trimethylation level)detected in an adjacent non-tumor tissue isolated from the subject,detected in a healthy tissue from a healthy subject, or established by apathologist with standard methods in the art.

By “sample” it means any biological sample derived from the subject,includes but is not limited to, cells, tissues samples and body fluids(including, but not limited to, mucus, blood, plasma, serum, urine,saliva, and semen).

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the inventionremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the invention can tolerate a wide variety offunctional groups; therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

Compounds of the present invention can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

Compounds of the present invention can be conveniently prepared by avariety of methods familiar to those skilled in the art. The compoundsof this invention with each of the formulae described herein may beprepared according to the following procedures from commerciallyavailable starting materials or starting materials which can be preparedusing literature procedures. These procedures show the preparation ofrepresentative compounds of this invention.

Compounds designed, selected and/or optimized by methods describedabove, once produced, can be characterized using a variety of assaysknown to those skilled in the art to determine whether the compoundshave biological activity. For example, the molecules can becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen themolecules described herein for activity, using techniques known in theart. General methodologies for performing high-throughput screening aredescribed, for example, in Devlin (1998) High Throughput Screening,Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays canuse one or more different assay techniques including, but not limitedto, those described below.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Recombinant Five-Component PRC2 Complex

Wild-type EZH2 (GenBank Accession No. NM_(—)004456) or Tyr641 mutantswere co-expressed with wild-type AEBP2 (GenBank Accession No.NM_(—)153207), EED (GenBank Accession No. NM_(—)003797), SUZ₁₂ (GenBankAccession No. NM_(—)015355) and RbAp48 (GenBank Accession No.NM_(—)005610) in Spodoptera frugiperda (Sf9) cells using a baculovirusexpression system. An N-terminal FLAG tag on the EED was used to purifyactive PRC2 complex from cell lysates (BPS Bioscience, catalog number51004). The purity of the final PRC2 preparations was assessed bySDS-PAGE with Coomassie blue staining.

Example 2 H3, H4 Peptide Panel

A library consisting of 44 peptides of 15 amino acids each wassynthesized by 21^(st) Century Biochemicals (Marlboro, Mass.). Thispeptide panel encompassed all of the amino acids of human histones H3and H4 with 5 residue overlaps between consecutive peptide sequences.The N-terminus of each peptide was appended with biotin, and theC-termini were represented as the amide. Purity (>95%) and identity wereconfirmed by liquid chromatography/mass spectral analysis.

For study of the H3-K27 methylation status dependence of enzymeactivity, peptides were synthesized representing the amino acid sequenceof human H3 from residues 21-44 (H3:21-44) with lysine 27 represented asthe unmodified, mono-methylated, di-methylated or tri-methylated sidechain amine. These peptides were purchased from New England Peptide(Gardner, Mass.) with biotin appended to the C-terminus of each peptide.

Example 3 Evaluation of H3-K27 Methylation Status in Cells

The cell lines OCI-LY19 (ACC 528), KARPAS-422 (ACC 32), and WSU-DLCL2(ACC 575) were obtained from DSMZ. The cell lines DB (CRL-2289) andSU-DHL6 (CRL-2959) were obtained from ATCC. OCI-LY19, WSU-DLCL2, and DBcell lines were grown in RPMI-1640 with 10% FBS, and KARPAS-422 andSU-DHL6 cell lines were grown in RPMI-1640 plus 20% FBS. Cells weregrown to a density of 1.5-2×10⁶ cells/mL and 1×10⁷ cells were harvestedby centrifugation at 264×g, washed in ice cold PBS and lysed byresuspension in a 10× pellet volume of RIPA lysis buffer containing 50mM Tris-HCl, 15 0 mM NaCl, 0.25% DOC, 1% NP-40, and 1 mM EDTA (Millipore#20-188), plus 0.1% SDS and protease inhibitor tablets (Roche #1836153).Lysates were sonicated by 2 rounds of 10 1-second bursts at setting 3with a Misonix XL-2000 to ensure efficient histone extraction, andcleared by centrifugation at 4° C. using a bench top centrifuge at14,000 rpm for 10 minutes. Protein concentration was determined by BCAassay (Pierce). Four micrograms of each lysate was fractionated on 4-20%Tris-Glycine gel (Invitrogen), transferred to PVDF, and probed with thefollowing antibodies in Odyssey blocking buffer: mouse anti-EZH2 (CST3147; 1:2000 dilution), rabbit anti-H3-K27me3 (CST 9733; 1:10000dilution), rabbit anti-H3-K27me2 (CST 9755; 1:5000 dilution), rabbitanti-H3-K27me1 (Active Motif 39377; 1:5000 dilution), and mouseanti-Total H3 (CST 3638; 1:20000 dilution). Following primary Abincubation, membranes were probed with IRDye 800CW donkey-anti-mouse IgG(LiCOR #926-32212) or Alexa Fluor 680 goat-anti-rabbit IgG (Invitrogen#A-21076) secondary Ab and imaged using the LiCOR Odyssey system.

Example 4 Enzymology

As noted above, it had previously been concluded that thedisease-associated changes at Tyr641 resulted in loss of function withrespect to EZH2-catalyzed H3-K27 methylation. However, a presumptivereduction in the rate of H3-K27 methylation due to'enzyme heterozygositywas difficult to rationalize as the basis for a malignant phenotype,especially in light of previous data indicating that overexpression ofEZH2, loss-of-function mutations in the corresponding H3-K27 demethylaseUTX, or overexpression of components of the PRC2, such as PHF19/PCL3,involved in increased H3-K27 trimethylation, all result in malignantphenotypes in specific human cancers. Morin et al. (2010) Nat Genet42:181-5; Martinez-Garcia et al. (2010) Nat Genet 42:100-1; Bracken etal. (2003) EMBO J 22:5323-35; Kleer et al. (2003) Proc Natl Acad Sci USA100:11606-11; Varambally et al. (2002) Nature 419:624-9; Simon et al.(2008) Mutat Res 647:21-9; van Haaften et al. (2009) Nat Genet.41:521-3; Wang et al. (2004) Gene 343:69-78; Cao et al. (2008) Mol CellBiol 28:1862-72; and Sarma et al. (2008) Mol Cell Biol 28:2718-31).Therefore, the enzymology of these mutations was explored in greaterdetail.

Recombinant PRC2 complexes were prepared with WT and Tyr641 mutantversions of human EZH2 (see Example 1 above; Cao et al. (2004) Mol Cell15:57-67). Equal concentrations (nominally 8 nM, based on proteindeterminations) of each complex were initially tested for the ability tocatalyze ³H-methyl transfer from labeled S-adenosyl methionine (SAM) toan unmodified peptide representing the amino acid sequence surroundingH3-K27 (H3:21-44) or to native avian erythrocyte oligonucleosomes. Aspreviously reported (Morin et al. (2010) Nat Genet 42:181-5), it wasfound that the WT enzyme displayed robust activity for methyl transferto this unmethylated peptidic substrate, but that none of the mutantenzymes displayed significant methyltransferase activity (FIG. 1A). Incontrast to the previously reported data and that in FIG. 1A, it wasfound that all of the mutant EZH2 constructs were activemethyltransferases against the avian nucleosome substrate (FIG. 1B). Thenucleosomes isolated from the avian natural source represent anadmixture of states of histone modification, including various states ofH3-K27 methylation as judged by Western blotting with H3-K27methylation-specific antibodies.

There are several potential explanations for the discordant activity ofthe mutant PRC2 complexes on peptide and nucleosome substrates. Onepossibility is that substrate recognition sites distal to the enzymeactive site (i.e., exosites) are important determinants of substratebinding and turnover; these sites would engage complementary recognitionelements on the nucleosome that are not available on small peptidicsubstrates. However, when E. coli-expressed, recombinant human histoneH3 was tested as a substrate for the WT and mutant PRC2 complexes, theresulting pattern of activity was identical to that seen for the peptidesubstrate; that is, the WT enzyme demonstrated robust methyltransferaseactivity against the H3 substrate, the Y641F mutant showed 7% theactivity of WT complex, and all other mutants displayed ≦1% the activityof WT complex. Hence, exosite engagement seems an unlikely explanationfor the current results. The nucleosome presents many lysine residuesbeyond H3-K27 as potential sites of methylation that would not bepresent in the small peptidic substrate. Thus, another possibility isthat mutation of Y641 alters the substrate specificity of EZH2 to resultin methylation of lysine residues other than H3-K27. This possibility isunlikely given the excellent agreement between mutant activity on smallpeptide and recombinant H3 protein substrates.

The apparent discordance between the present results and thosepreviously reported was resolved when the enzymatic activity of the WTand mutant PRC2 complexes were tested against a panel of peptidicsubstrates that represent all possible lysine (K) residues of histone H3and histone H4 (see Example 2 above). All of the enzyme forms showedsignificant activity only against peptides containing the equivalent ofresidue H3-K27. The specific activity of the mutants, however, wasgreatly reduced relative to WT in the order WT>>Y641F>Y641S-Y641H>Y641N,again consistent with previous reported findings.

Example 5 Enzymology

To understand further the enzymatic activity of these mutants, and toreconcile the apparent discrepancy between activity against peptidic andnucleosome substrates, the ability of the enzyme forms to catalyzefurther methylation of various H3-K27 methylation states in the contextof the H3:21-44 peptide was studied. As stated above, it was found thatall of the mutant enzymes were deficient catalysts of unmodified H3-K27peptide methylation, relative to the WT enzyme. Remarkably, however, allof the mutant enzymes were found to be superior to WT enzyme incatalyzing further methylation of the mono- and especially thedi-methylated H3-K27 peptides (FIG. 2). Thus, the data suggest that theWT enzyme is most efficient in catalyzing the zero- to mono-methylationreaction. The mutant enzymes are defective in catalyzing this initialstep, but are more efficient than the WT enzyme in catalyzing thesubsequent steps leading from mono-methyl to di- and tri-methyl H3-K27.

The origins of the differential substrate specificities of WT and mutantEZH2 were explored through steady state enzyme kinetics. As summarizedin Table 2, the mutations have minimal effects on ground-state substraterecognition, as demonstrated by the similar values of K_(m) fornucleosome and of K_(1/2) for peptide substrates. In all cases thepeptidic substrates displayed sigmoidal binding behavior; hence theconcentration of peptide resulting in half-maximal velocity is reportedhere as K_(1/2) instead of the more common Michaelis constant, K_(m).Copeland (2005) Evaluation of Enzyme Inhibitors in Drug Discovery: AGuide to Medicinal Chemists and Pharmacologists, Wiley. The SAM K_(m)likewise displayed minimal variation among the enzyme forms, rangingfrom 208±50 to 304±64 nM. Instead, the differences in substrateutilization appear to have their origin in transition state recognition,as demonstrated by differences in k_(cat) values among the enzymes forvarious substrates (Table 2). As a result, the catalytic efficiency,quantified as the ratio k_(cat)/K (where K is either K_(m) or K_(1/2),depending on substrate identity; vide supra), varies between the WT andmutant enzymes for different states of H3-K27 methylation (Table 2).

TABLE 2 Steady state kinetic parameters for methylation reactionscatalyzed by PRC2 containing wild-type or Y641 mutants of EZH2.Substrate H3-K27 Methylation K k_(cat) k_(cat)/K Enzyme Status (nM) (h⁻¹× 10⁻²) (h⁻¹ · nM⁻¹ × 10⁻⁴) WT 0 184 ± 10 84.0 ± 3.0 45.7 ± 3.0 1 436 ±42 65.4 ± 5.8 15.0 ± 2.0 2 178 ± 16  6.0 ± 0.3  3.4 ± 0.3 Nucleosome 141± 31 42.6 ± 2.6 30.2 ± 6.9 Y641F 0 240 ± 19  4.8 ± 0.3  2.0 ± 0.2 1  404± 124 15.0 ± 4.3  3.7 ± 1.6 2 191 ± 10 84.0 ± 2.8 44.0 ± 2.7 Nucleosome176 ± 19 65.4 ± 2.0 37.2 ± 4.2 Y641H 0 —^(a) — — 1 319 ± 57 28.2 ± 3.7 8.8 ± 2.0 2 148 ± 9  22.8 ± 0.9 15.4 ± 1.1 Nucleosome 140 ± 22 23.4 ±1.0 16.7 ± 2.7 Y641N 0 — — — 1 280 ± 11 23.4 ± 0.8  8.4 ± 0.4 2 157 ± 1196.0 ± 4.0 61.1 ± 5.0 Nucleosome 191 ± 34 23.4 ± 1.3 12.3 ± 2.3 Y641S 0— — — 1 249 ± 8  27.6 ± 0.8 11.1 ± 0.5 2 136 ± 8  59.4 ± 2.0 43.7 ± 3.0Nucleosome 137 ± 28 23.4 ± 1.4 17.1 ± 3.6 ^(a)Activity too low tomeasure.

Example 6 Enzymology

The steady state kinetic parameters listed in Table 2 made it possibleto calculate the , expected levels of different H3-K27 methylationstates for cells heterozygous for the various mutant EZH2 forms,relative to cells homozygous for the WT enzyme. To perform thesesimulations, a number of simplifying assumptions were made: (1) thatsteady state enzyme kinetics are relevant to PRC2-catalyzed H3-K27methylation in the cellular context and that all measurements are madeat the same time point in cell growth; (2) that the mutant and WT enzymeare expressed at equal levels in heterozygous cells and that the totalEZH2 level is equal in all cells; (3) that the cellular concentration ofSAM, relative to its K_(m) is saturating and does not change among thecells; (4) that the cellular concentration of nucleosome, is similar toits K_(m) and likewise does not change among cells; (5) that EZH1catalyzed methylation of H3-K27 was insignificant and constant among thecells; and (6) that any H3-K27 demethylase activity was also constantamong the cells.

With these assumptions in place, the predictions illustrated in FIG. 3Awere obtained for relative levels of H3-K27me3 (top panel), H3-K27me2(middle panel) and H3-K27me1 (bottom panel). A clear pattern emergesfrom these simulations. The level of H3-K27me3 increases relative to WTcells for all mutant-harboring cells, ranging from a 30% increase forthe Y641H mutant to >400% for the Y641N mutant. At the same time, thelevels of H3-K27me2 decreases to <50% of WT for all of the mutants, andthe levels of H3-K27me1 are reduced by approximately half for allmutants, relative to WT.

The relative levels of the H3-K27 methylation states in B-cell lymphomacell lines that are known to be homozygous for WT EZH2 (OCI-LY19) orheterozygous for EZH2 Y641N (DB, KARPAS 422, and SU-DHL-6) or EZH2 Y641F(WSU-DLCL2) were then measured by Western blotting (FIG. 3B). Thepattern of relative H3-K27 methylation states seen in FIG. 3 b is inexcellent agreement with the results of the simulations based on invitro steady state kinetic parameters, despite the assumptions used inthe simulations and the use of a non-physiological peptide surrogate assubstrate.

Thus, increased H3-K27me3 was observed for all Y641 mutant-harboringcells relative to WT, decreased H3-K27me2 was observed for all Y641mutant-harboring cells relative to WT, and decreased H3-K27me1 wasobserved for at least two of the four mutant cell lines. Thenear-comparable levels of H3-K27me1 in WT and KARPAS 422 and SU-DHL-6cells may reflect different expression levels of WT and mutant EZH2,different contributions of EZH1, or other factors not accounted for inthe simulations. Nevertheless, the concordance between the predicted andexperimental patterns of H3-K27 methylation status is remarkable andsupports the view that enzymatic coupling between WT and mutant EZH2leads to increased H3-K27me3, thus resulting in the malignant phenotypeof cells that are heterozygous for these mutants.

Example 7 In Vitro Assays of PRC2 Methyltransferase Activity

Flashplate Assay with Peptide Substrate.

For initial comparison of WT and Y641 mutants of EZH2, biotinylatedhistone H3:21-44 peptide containing unmethylated K27 (New EnglandPeptide), monomethylated K27 (Millipore) or dimethylated K27 (Millipore)at a concentration of 800 nM was combined with a mixture ofS-adenosylmethionine-Cl (SAM) at 1,700 nM, and 300 nM tritiated SAM(Perkin Elmer). This substrate combination was then added to the PRC2 inassay buffer (20 mM BICINE, 1 mM DTT, 0.002% Tween 20, 0.005% bovineskin gelatin (BSG), pH 7.6). Reactions were allowed to proceed for theindicated time interval and then quenched by addition of excess cold SAM(600 μM final concentration). Quenched reaction mixtures weretransferred to a streptavidin-coated Flashplate (Perkin Elmer, catalognumber SMP410), allowed to bind for one hour, and then detected on aTopCount NXT HTS scintillation and luminescence counter (Perkin Elmer).Each time point represented the average of six individual reactions.Steady state kinetic parameters were determined under identical reactionconditions except that the concentration of peptide or SAM was varied,while at saturating conditions of the other substrate. Velocity wasplotted as a function of varied substrate concentration and the datawere fitted to the untransformed version of the Michaelis-Mentenequation or the untransformed version of a sigmoidal kinetic equation tocalculate values of K and k_(cat). Standard errors of fitted parametersare listed in Table 2 and were used to construct the error barsillustrated in FIG. 2 panels B and C. Error associated with k_(cat)/K(Table 2) were calculated according to standard methods of errorpropagation; the fractional error of k_(cat)/K was determined as:

$\begin{matrix}{{\mu \frac{k_{cat}}{K}} = \sqrt{\left( \frac{\mu \; k_{cat}}{k_{cat}} \right)^{2} + \left( \frac{\mu \; K}{K} \right)^{2}}} & (1)\end{matrix}$

where μk_(cat) is the standard error of k_(cat) and μK is the standarderror of K.

Filterplate Assay with Oligonucleosome.

Chicken erythrocyte oligonucleosomes were purified as previouslydescribed. Fang et al. (2004) Methods Enzymol 377:213-26. Nucleosomeswere combined with a mixture of SAM and tritiated SAM, and added to PRC2in assay buffer (20 mM BICINE, 100 mM KCl, 1 mM DTT, 0.002% Tween 20,0.005% BSG, pH 7.6). Reactions were run and quenched as above. Quenchedreaction mixture was transferred to a glass fiber filterplate(Millipore, catalog number MSFBN6B) and washed three times with 10%trichloroacetic acid and allowed to dry. Microscint Zero (30 μL) wasadded and tritium incorporation was detected on a TopCount scintillationand luminescence counter. Steady state parameters were determined underidentical reaction conditions except that the concentration ofnucleosome or SAM was varied while at saturating conditions of the othersubstrate. Velocity was plotted as a function of varied substrateconcentration and fitted to the untransformed version of theMichaelis-Menten equation to derive the values of K_(m) and k_(cat) asdescribed above.

Example 8 Compound Preparation A. Preparation of Compound 63

To a solution of9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(Townsend, A. P. et al. (2009) Org. Let. 11:2976-2979) (3.05 g, 9.96mmol) in DCE (250 mL) was added (9H-fluoren-9-yl)methyl(2-oxoethyl)carbamate (2.8 g, 9.96 mmol) and NaB(OAc)₃H (2.96 g, 13.95mmol), the mixture stirred for 4 h at room temperature. K₂CO₃ solutionwas added to pH at 8-9. DCM was added, the organic layer was dried withNa₂SO4, concentrated and purified by SGC (DCM:MeOH=30:1) to give 63 (2.9g, yield: 50.9%).

B. Preparation of Compound 65

To a solution of 63 (2.9 g, 5.08 mmol) in DCE (250 mL), (S)-benzyl2-((tert-butoxycarbonyl)amino)-4-oxobutanoate (1.56 g, 5.08 mmol) andNaB(OAc)₃H (1.51 g, 7.11 mmol) were added, the mixture stirred for 4 hat room temperature. K₂CO₃ solution was added to pH at 8-9. DCM wasadded, the organic layer was dried with Na₂SO₄, concentrated andpurified with SGC (DCM: MeOH=100:1) to give 65 (2.8 g, yield: 63.9%).

C. Preparation of Compound 75

Step 1. To a solution of 65B (2.2 g, 2.55 mmol) in DCM (10 mL), Et₂NH(1.1 g, 15.3 mmol) were added, the mixture stirred for 4 h at roomtemperature. The mixture was concentrated to give crude 72 (2.2 g).

Step 2. To a stirred solution of 72 (167 mg, 0.26 mmol) in MeOH (4 mL),2-(4-chlorophenyl)acetaldehyde (40 mg, 0.26 mmol) was added and stirredat room temperature for 20 min. Then Na(OAc)₃BH (83 mg, 0.39 mmol) andHOAc (0.4 mL) was added and stirred overnight. Then NaHCO₃ (aq) wasadded and extracted with DCM (25 mL×3), washed with brine, dried withNa₂SO₄ and concentrated. The crude product was purified by preparativeTLC (DCM/MeOH=10:1) to afford 73 (30 mg, yield: 14%) as white powder.LC/MS (m/z): 779.7 [M+1]⁺.

Step 3. A mixture of 73 (30 mg, 0.038 mmol) and 10% Pd/C (15 mg) in MeOH(2 mL) was stirred at room temperature under H₂ overnight. The mixturewas filtered and the filtrate was concentrated to give crude product.The crude product was purified by preparative TLC (DCM/MeOH=8:1) toafford 74 (20 mg, yield: 69%) as white powder. LC/MS (m/z): 689.7[M+1]⁺.

Step 4. A solution of 74 (20 mg, 0.028 mmol) in 90% TFA (1 mL) wasstirred at room temperature for 1 h, and concentrated as a solid toremove. TFA to give the compound 75 (TFA salt) as a colorless oilwithout purification. LC/MS (m/z): 549.7 [M+1]⁺.

Example 9 Inhibition of EZH2 Wild-Type and Y641 Mutants by SAH

S-Adenosyl-L-homocysteine (SAH) was serially diluted 3 fold in DMSO for10 points and 1 μL was plated in a 384 well microtiter plate. Positivecontrol (100% inhibition standard) was 100 μM final concentration of SAHand negative control (0% inhibition standard) contained 1 μL of DMSO.SAH was then incubated for 30 minutes with 40 μL per well of EZH2wild-type and mutants at 8 nM in pH 7.6 assay buffer (20 mM BICINE, 100mM KCl, 1 mM DTT, 0.002% Tween 20, 0.005% BSG). A substrate mix at 10 μLper well was added which contained S-adenosylmethionine-C1 (SAM) at 150nM and tritiated SAM at 100 nM, and biotinylated oligonucleosome at 150nM in pH 7.6 assay buffer. Quenched enzyme reaction was transferred to astreptavidin-coated Flashplate (Perkin Elmer, catalog number SMP410),allowed to bind for one hour, and detected on a TopCount NXT HTS (PerkinElmer).

Results are shown in FIG. 7. IC50 values are shown in Table 3.

TABLE 3 Inhibition of WT EZH2 and Y641 mutants of EZH2 by SAH. WT Y641HY641S Y641N Y641F IC50, 0.467 0.263 0.283 0.380 4.80 μM

Example 10 Inhibition of EZH2 Wild-Type and Y641 Mutants by Compound 75

Compound 75 was serially diluted 3 fold in DMSO for 10 points and 1 μLwas plated in a 384 well microtiter plate. Positive control (100%inhibition standard) was 100 μM final concentration of SAH and negativecontrol (0% inhibition standard) contained 1 μL of DMSO. Compound 75 wasthen incubated for 30 minutes with 40 μL per well of EZH2 wild-type andmutants at 8 nM in pH 7.6 assay buffer (20 mM BICINE, 100 mM KCl, 1 mMDTT, 0.002% Tween 20, 0.005% BSG). A substrate mix at 10 μL per well wasadded which contained S-adenosylmethionine-Cl (SAM) at 150 nM andtritiated SAM at 100 nM, and biotinylated oligonucleosome at 150 nM inpH 7.6 assay buffer. Quenched enzyme reaction was transferred to astreptavidin-coated Flashplate (Perkin Elmer, catalog number SMP410),allowed to bind for one hour, and detected on a TopCount NXT HTS (PerkinElmer).

Results are shown in FIG. 8. IC50 values are shown in Table 4.

TABLE 4 Inhibition of WT EZH2 and Y641 mutants of EZH2 by Compound 75.WT Y641S Y641N Y641F Y641H IC50, 8.95 2.50 4.10 7.18 7.56 μM

Example 11 H3-K27me2/me3 Ratios Predict Sensitivity to an EZH2 Inhibitor

Tumor cell lines heterozygous for the EZH2 (Y641) mutation displayincreased levels of H3-K27me3, the methylation state of H3-K27 thoughtto be important in tumorigenesis. Levels of the mono (H3-K27me1), di(H3-K27me2), or trimethylated (H3-K27me3) forms of H3-K27 in a panel ofcell lines that were WT for EZH2, or heterozygous for EZH2 (Y641)mutations were evaluated. Cell lines used are listed in Table 5. Themajority of lines are B-cell lymphoma lines, however two melanoma lineswere also included. IGR1 is a melanoma line that has recently been foundto contain a Y641N mutation in EZH2, and A375 cells were included as aWT EZH2 melanoma control line. FIGS. 9A and B show the results ofwestern blot analysis of histones isolated from this cell line panelprobed with antibodies recognizing H3-K27me1, H3-K27me2, or H3-K27me3.In general, global H3-K27me3 levels are higher in Y641 mutant containingcell lines than in cell lines expressing WT EZH2 exclusively. Theexception is Farage cells, where H3-K27me3 levels were similar to thosein WT lines. More striking are the dramatically lower levels ofH3-K27me2 in EZH2 Y641 mutant cell lines relative to wild type celllines. Little or no H3-K27me2 signal was observed in western blot ofhistones extracted from Y641 mutant cell lines, whereas the signalobserved with the same antibody in WT cell lines was more intense thanthat observed with the antibody specific for H3-K27me3. Overall, in WTcell lines the western blot signal with an HK27me2 antibody was higherthan the signal observed with the H3-K27me3 antibody, whereas theopposite was true in Y641 mutant cell lines. Thus the ratio ofH3-K27me3/me2 signal in Y641 lines is higher than that observed in WTlines.

The H3-K27 methylation state can also be examined by Mass spectrometry(MS), an independent method that does not rely on antibody reagents. TheMS analysis demonstrated that H3-K27me3 levels are higher in Y641 mutantand Pfeiffer lines (A677G) than in the other WT lines, whereas theopposite is true for H3-K27me2 levels. In the Y641 mutant and Pfeifferlines (A677G), H3-K27me3 levels were higher than H3-K27me2 levels,whereas the opposite was true in the other WT lines. These results areconsistent with those observed by western blot analysis in FIGS. 9A andB.

The differences in H3-K27 methylation state was also detected byimmunocytochemistry using antibodies to H3-K27me2 or H3-K27me3. Thisimmunohistochemistry assay is used for detecting aberrant H3-K27me2/3ratios associated with Y641 mutant EZH2 in formalin fixed paraffinembedded patient tumor tissue samples. A panel of five WT and five Y641mutant lymphoma cell line pellets were fixed and embedded in paraffinblocks and stained with anti-H3-K27me2 or H3-K27me3 antibodies. Anantibody to histone H3 was included as a positive control, since allcells should contain nuclear histone H3. FIG. 10 shows that all celllines were positive in 100% of cells for both H3-K27me3 and H3 staining.Under these conditions, no clear difference in H3-K27me3 stainingintensity was observed between WT and Y641 mutant cell lines. This mayreflect the limited dynamic range of chromogenic immunocytochemistrystaining compared to other methods of detection. However, as shown inFIG. 11, cell lines could be clearly segregated into those stainingpositive or negative for H3-K27me2. All WT cell lines stained positivefor H3-K27me2, whereas all Y641 mutant cell lines and Pfeiffer cells(A677G) showed no staining with the H3-K27me2 antibody. These resultsare consistent with those obtained by western and MS analysis.

Without wishing to be bound by theory, the increased levels of H3-K27me3associated with the gain of function EZH2 (Y641) mutations may rendercells bearing EZH2 mutations more sensitive to small molecule EZH2inhibitors. To evaluate whether the increased H3-K27me3 and/or decreasedH3-K27me2 levels observed in Pfeiffer cells in the absence of an EZH2Y641 mutation would also correlate with sensitivity to EZH2 inhibitors,two compounds that demonstrate potent inhibition of EZH2 in biochemicalassays with IC50s of 85 and 16 nM respectively were tested. Treatment ofWSU-DLCL2 cells with either compound led to inhibition of globalH3-K27me3 levels, confirming their ability to enter cells and inhibitcellular EZH2 methyltransferase activity (FIG. 12).

The sensitivity of a panel of WT and Y641 mutant cell lines to eachcompound was evaluated in proliferation assays. Because theanti-proliferative activity of EZH2 inhibitors takes several days tomanifest, compounds were assessed in 11-day proliferation assays. FIG.13 shows representative growth curves for WT (OCI-LY19), or Y641 mutant(WSU-DLCL2) cell lines treated with the test compounds. Both compoundsdemonstrated anti-proliferative activity against WSU-DLCL2 cells, butlittle activity against OCI-LY19 cells. Inhibitor A was a more potentinhibitor of WSU-DLCL2 proliferation than Inhibitor B and this isconsistent with Inhibitor A being a more potent inhibitor of EZH2 inbiochemical assays. Proliferation assays were performed in a panel of WTand Y641 mutant lymphoma cell lines, with Inhibitor B, and day 11 IC90values were derived. FIG. 14A shows IC90 values of lymphoma cell linesgrouped by EZH2 Y641 status. Overall, Y641 mutant cell linesdemonstrated increased sensitivity to EZH2 inhibitors relative to WTcell lines, although RL and SUDHL4 cells were significantly lesssensitive than other mutant lines. Pfeiffer cells (A677G) demonstratehigh H3-K27me3 and low H3-K27me2 levels, and so grouping cell linesaccording to high H3-K27me3 and low H3-K27me2 gives betterdiscrimination of EZH2 inhibitor sensitivity as shown for Inhibitor B inFIG. 14B. Thus, high H3-K27me3 and low H3-K27me2 levels can be used topredict sensitivity to EZH2 inhibitors, independent of knowledge ofmutational status.

These results demonstrates that identifying EZH2 Y641 mutations inpatient tumors and/or detecting low levels of H3-K27me2 relative toH3-K27me3 through use of techniques such as western blot, MS or IHC in apatient can be used to identify which patient will respond to EZH2inhibitor treatment.

TABLE 5 Cell lines used in this study. Cancer EZH2 Status Cell LineLymphoma: Wild Type OCI-LY19 DLBCL (Diffuse Large HT Cell B CellLymphoma) MC116 and other B-cell BC-1 Lymphoma BC-3 Toledo DOHH-2 FarageSR NU-DHL-1 NU-DUL-1 Y641 Mutation SU-DHL-10 (Y641F) DB (Y641N) KARPAS422 (Y641N) SU-DHL-6 (Y641N) WSU-DLCL-2 (Y641F) RL (Y641N) SU-DHL-4(Y641S) Melanoma Wild Type A375 Y641 Mutation IGR-1 (Y641N)

Example 12 Recombinant 4-Component PRC2 Complexes

Wild-type (WT) EZH2 (GenBank Accession No. NM_(—)004456) or A677G andA687V mutants were co-expressed with wild-type EED (GenBank AccessionNo. NM_(—)003797), SUZ12 (GenBank Accession No. NM_(—)015355) and RbAp48(GenBank Accession No. NM005610) in Spodoptera frugiperda (Sf9) cellsusing a baculovirus expression system. An N-terminal FLAG tag on the EEDwas used to purify active PRC2 complex from cell lysates. The purity ofthe final PRC2 preparations was assessed by SDS-PAGE with Coomassie bluestaining and protein concentration was determined using a bovine serumalbumin standard curve in a Bradford assay.

Example 13 In Vitro Assays of PRC2 Methyltransferase Activity

Standard Procedure for Flashplate Assay with Peptide Substrates.

Activity of the wild-type or mutant EZH2-containing PRC2 complexes wasinvestigated using a series of four peptides representing the amino acidsequence of human H3 from residues 21-44 (H3:21-44) with lysine 27represented as the unmodified, monomethylated, dimethylated ortrimethylated side chain amine, consisting of the following sequence,with the H3-K27 lysine subject to modification underlined,ATKAARKSAPATGGVKKPHRYRPGG[K-Ahx-Biot]-amide (SEQ ID NO: 20). Biotin(Biot) was appended to a C-terminal lysine (K) residue through anaminohexyl linker (AHX) attached to the lysine side chain amine (21^(st)Century Biochemicals). For comparison of mutant of WT and mutant EZH2activity, biotinylated histone H3:21-44 peptides were combined with amixture of S-adenosylmethionine (SAM; Sigma-Aldrich) and tritiated SAM(³H-SAM; American Radiolabeled Chemicals) and recombinant 4-componentPRC2 in assay buffer (20 mM BICINE, 1 mM DTT, 0.002% Tween 20, 0.005%bovine skin gelatin (BSG), pH 7.6). Reactions were allowed to proceedfor the indicated time interval and then quenched by addition of excesscold SAM (100 μM final concentration). Quenched reaction mixtures weretransferred to a streptavidin-coated Flashplate (Perkin Elmer, catalognumber SMP410), and allowed to bind for one hour before the plates werewashed in a Biotek EL-405× platewasher and read on a TopCount NXT HTSscintillation and luminescence counter (PerkinElmer).

Example 14 Enzymology

Recombinant 4-component PRC2 complexes were prepared with wild-type andeither A677G or A687V mutant versions of EZH2 (see Example 12 above; Caoet al. (2004) Mol Cell 15:57-67). Each complex was initially tested forthe ability to catalyze ³H-methyl transfer from labeled S-adenosylmethionine (SAM) to each of the four H3:21-44 peptides. Enzyme wasserially diluted, and a mixture of peptide (200 nM) and SAM (200 nM³H-SAM and 800 nM unlabeled SAM) was added. Reactions were quenched at15 minute intervals by the addition of an excess of unlabeled SAM andreaction velocity was calculated based on the linear regression of rawcounts per minute (CPM) vs. time. As shown in FIG. 15, the mutantenzymes displayed a different pattern of activity than the wild-typeenzyme. The A677G and A687V mutants had robust activity on all of theunmethylated, monomethylated and dimethylated H3:21-44 peptides, whereasthe wild-type enzyme only showed robust activity on the unmethylated andmonomethylated peptides. The control peptide, containing fullytrimethylated H3-K27 was not methylated in the assay, indicating thatH3-K27 was the target lysine.

Example 15 Enzymology

To further understand the enzymatic activity of these mutants, theorigins of the differential substrate specificities of wild-type andmutant EZH2 were explored through steady-state enzyme kinetics.Reactions containing a titration of the H3-K27 peptides with fixedenzyme (4 nM) and SAM (200 nM ³H-SAM and 800 nM unlabeled SAM) wereperformed. In all cases the peptidic substrates displayed sigmoidalbinding behavior; hence the concentration of peptide resulting inhalf-maximal velocity is reported here as K_(1/2) instead of the morecommon Michaelis-Menten constant, K_(m) (Copeland (2005) Evaluation ofEnzyme Inhibitors in Drug Discovery: A Guide to Medicinal Chemists andPharmacologists, Wiley). As summarized in Table 6, the mutations have aneffect on ground-state substrate recognition as demonstrated by lowerK_(1/2) for the unmethylated peptide substrates and higher K_(1/2)values for the dimethylated peptide substrates. Additionally, themaximal velocities of the enzymes are affected by the mutations. TheA677G mutation leads to 2.9-, 3.7- and 22-fold increases in k_(cat) onrespective un-, mono-, and dimethyl H3-K27 peptide substrates, while theA687V mutation results in a 3-fold reduction in k_(cat) on the unmethylH3-K27 peptide, but produces respective 3.5- and 2.5-fold increases ink_(cat) on the mono- and dimethyl H3-K27 peptides. The SAM K_(m)displayed minimal variation among the enzyme forms, 403±64 nM to 899±89nM on the substrate containing the preferred methylation state at theH3-K27 residue.

TABLE 6 Summary of Steady-State Enzyme Kinetics for WT and Mutant EZH2Enzymes Peptide Substrate H3- K27 k_(cat)/K_(1/2) Methylation (h⁻¹ ·Enzyme Status K_(1/2) (nM) k_(cat) (h⁻¹) nM⁻¹ × 10⁻⁴) *WT 0 154 ± 124.80 ± 0.20 305 ± 26 1 337 ± 26 3.33 ± 0.21 99 ± 9 2 144 ± 11 1.08 ±0.04 75 ± 6 A677G 0 88 ± 6 14.05 ± 0.54  1590 ± 120 1 222 ± 51 12.25 ±1.67   570 ± 150 2  522 ± 117 23.85 ± 3.60   450 ± 120 A687V 0 43 ± 31.58 ± 0.08 370 ± 30 1 176 ± 13 11.49 ± 0.50  650 ± 60 2  352 ± 155 2.70± 0.65  80 ± 40 *Wild-type data was previously published in Wigle etal., Febs Lett (2011) Oct 3; 585(19): 3011-4 using 4-component EZH2.

Example 16 Inhibition of Wild-Type EZH2 and EZH2 Mutants by EZH2Inhibitors

Test compounds were serially diluted 3-fold in DMSO in a 10 point-curveand 1 μL was spotted into a 384-well microplate in duplicate using aPlatemate Plus equipped with 384-channel head (Thermo Scientific). Thefinal top concentration of test compounds in the assay was 10 μM.Positive control (100% inhibition standard) was 1 mM final concentrationof SAH and negative control (0% inhibition standard) contained 1 μL ofDMSO. Test compounds were then incubated for 30 minutes with 40 μl, perwell of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (finalconcentration was 0.1 nM) and A677G and A687V EZH2 (for each, finalconcentration was 2 nM) and peptide in 1× assay buffer (20 mM BICINEpH=7.6, 1 mM DTT, 0.002% Tween 20, 0.005% BSG). For the wild-type EZH2and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylatedK27 was present at a final concentration of 200 nM, while in the A687VEZH2 assay, biotinylated peptide H3:21-44 with monomethylated K27 waspresent at a final concentration of 200 nM and in the Y641F EZH2 assaybiotinylated peptide H3:21-44 with dimethylated K27 was present at afinal concentration of 200 nM. To initiate the reaction containing thewild-type EZH2 enzyme, a substrate mix of 10 μL per well was added thatcontained unlabeled SAM (final concentration was 1800 nM) and ³H-SAM(final concentration was 200 nM) in 1× assay buffer. To initiate thereaction containing the Y641F EZH2 enzyme a substrate mix of 10 μL perwell was added that contained unlabeled SAM (final concentration was 700nM), and ³H-SAM (final concentration was 300 nM) in 1× assay buffer. Toinitiate the reactions containing the A677G or A687V EZH2, a substratemix of 10 μL per well was added which contained unlabeled SAM (finalconcentration was 400 nM) and ³H-SAM (final concentration was 100 nM).Reactions proceeded for 90 min, then were quenched with excess unlabeledSAM (167 μM), then were transferred to a streptavidin-coated Flashplate(PerkinElmer, catalog number SMP410), allowed to bind for one hour, anddetected on a TopCount NXT HTS (PerkinElmer). The IC₅₀ values areobtained from 4-parameter fits of the % inhibition of enzyme activityand are tabulated in Table 7. The formulae used to derive IC₅₀ valuesare indicated below.

% Inhibition Calculation

${\% \mspace{14mu} {inh}} = {100 - {\left( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} \right) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter 1050 Fit

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\left( {1 + \left( \frac{X}{{IC}_{50}} \right)^{{Hill}\; {Coefficient}}} \right.}}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

TABLE 7 Inhibition of wild-type and mutant EZH2 by EZH2 inhibitorsCompound EZH2 Y641F A677G A687V Number Inhibitor WT (uM) (uM) (uM)(uM) 1. SAH 6.9082 16.6193 6.2379 5.9034 2. EPZ004710 2.9758 3.78870.3187 0.5378 3. EPZ004744 1.5203 0.7432 0.1128 0.1354 4. EPZ0050300.2600 0.1846 0.0418 0.0504 5. EPZ005100 0.3579 0.2923 0.0316 0.0599 6.EPZ005260 2.3755 1.5781 0.2130 0.3189 7. EPZ005687 0.0950 0.0750 0.01130.0082 8. EPZ006089 0.2300 0.3110 0.0190 0.0277 9. EPZ006438 0.00620.0111 0.0022 0.0015 10. EPZ006632 0.0025 0.0034 0.0040 0.0032 11.EPZ007038 0.0099 0.0123 0.0060 0.0032 12. EPZ007209 0.0065 0.0074 0.00860.0068 13. EPZ007210 0.0043 0.0044 0.0038 0.0036 14. EPZ007227 0.01430.0207 0.0122 0.0128 15. EPZ007426 0.0181 0.0088 0.0034 0.0040 16.EPZ007428 0.0014 0.0055 0.0019 0.0021 17. EPZ007478 0.0088 0.0114 0.00420.0071 18. EPZ007648 0.0025 0.0079 0.0058 0.0067 19. EPZ007649 0.00940.0092 0.0096 0.0082 20. EPZ007655 0.0125 0.0104 0.0192 0.0171 21.EPZ007692 0.0100 0.0117 0.0116 0.0103 22. EPZ007789 0.0108 0.0114 0.00480.0051 23. EPZ007790 0.0169 0.0158 0.0073 0.0065 24. EPZ008205 0.01290.0093 0.0112 0.0101 25. EPZ008277 0.0333 0.0092 0.0023 0.0055 26.EPZ008278 0.0384 0.0106 0.0093 0.0191 27. EPZ008279 0.0223 0.0182 0.00220.0051 28. EPZ008280 0.0067 0.0029 0.0028 0.0039 29. EPZ008286 0.00430.0025 0.0015 0.0018 30. EPZ008335 0.0065 0.0033 0.0015 0.0029 31.EPZ008336 0.0057 0.0036 0.0013 0.0024 32. EPZ008337 0.0087 0.0015 0.00080.0014 33. EPZ008338 0.0120 0.0096 0.0031 0.0072 34. EPZ008344 0.01240.0036 0.0016 0.0046 35. EPZ008491 0.0091 0.0014 0.0029 0.0029 36.EPZ008493 0.1320 0.0127 0.0882 37. EPZ008494 0.0079 0.0046 0.0028 0.003838. EPZ008495 0.0134 0.0104 0.0063 0.0084 39. EPZ008496 0.0154 0.01040.0040 0.0076 40. EPZ008497 >10.0 uM 3.2876 0.9735 1.2184 41. EPZ0085920.0145 0.0051 0.0035 0.0075 42. EPZ008623 0.2440 0.1835 0.0922 0.106743. EPZ008630 0.0034 0.0029 0.0035 0.0032 44. EPZ008681 0.0029 0.00150.0027 0.0047 45. EPZ008686 0.0073 0.0055 0.0045 0.0096 46. EPZ0089890.0094 0.0069 0.0022 0.0028 47. EPZ008990 0.0061 0.0067 0.0016 0.001748. EPZ008991 0.0348 0.0293 0.0094 0.0193 49. EPZ008992 0.3333 0.16780.0638 0.1188 50. EPZ008994 0.0715 0.0275 0.0121 0.0205 51. EPZ0090900.0300 0.0111 0.0120 0.0227 52. EPZ009097 0.0047 0.0039 0.0046 0.003653. EPZ009099 0.0765 0.0255 0.0057 0.0222 54. EPZ009152 0.0030 0.00310.0029 0.0033 55. EPZ009153 0.0052 0.0032 0.0056 0.0037 56. EPZ0091540.0278 0.0420 0.0141 0.0438 57. EPZ009155 0.0563 0.0524 0.0251 0.062358. EPZ009156 0.0034 0.0217 0.0051 0.0132 59. EPZ009157 0.0397 0.04430.0214 0.0436 60. EPZ009158 0.0021 0.0016 0.0027 0.0027 61. EPZ0091610.0009 0.0008 0.0011 62. EPZ009162 0.0657 0.0351 0.0146 0.0178

Example 17 A677 Mutant Shows the Highest Sensitivity to the EZH2Inhibitors

Pfeiffer cells were obtained from ATCC(CRL-2632). WSU-DLCL2 (ACC 575)and OCI-Ly19 (ACC 528) cells were obtained from DSMZ. All cells weremaintained in RPMI+10% FBS. For cell proliferation analysis,exponentially growing Pfeiffer, WSU-DLC2, or OCI-Ly19 cells were plated,in triplicate, in 96-well plates at a density of 1×10⁵ cells/mL, 5×10⁴cells/mL, or 2.5×10⁵ cells/mL (respectively) in a final volume of 150uL. Cells were incubated with Compound #7 at final concentrationsranging from 0.011 to 25 uM for IC₅₀ determination over an 11-day timecourse. Cells were incubated with Compound #13 at final concentrationsranging from 0.00004 to 10 uM for IC₅₀ determination over an 11-day timecourse. Every 3-4 days, viable cell numbers were determined using theGuava Viacount assay (Millipore #4000-0040) and analyzed on a GuavaEasyCyte Plus instrument. After cell counts, growth media and EPZ2inhibitor (Compound #7 or Compound #13) were replaced, and cells weresplit back to the original plating density. The final split-adjustednumber of viable cells/mL from day 11 of the time course was used tocalculate the proliferation IC₅₀ values, using Graphpad Prism software.

The Pfeiffer cell line, containing the heterozygous EZH2 mutation A677G,is shown to be sensitive to EZH2 inhibition by a small moleculeinhibitor Compound #7. Proliferation inhibition is seen as early as 96hr post-inhibitor treatment. The proliferation IC₅₀ after 11 days forPfeiffer cells treated with Compound #7 is 5.2 nM, compared to WSU-DLCL2cells, which contain the Y641F heterozygous mutation, and have an IC₅₀of 270 nM or OCI-Ly19 cells, which are WT for EZH2, and have an IC₅₀ of3000 nM. These results show that in a cellular context, the sensitivityof WT and mutant EZH2 to inhibition by small molecule inhibitor isA677G>>>Y641F>>WT, as measured by proliferation.

The Pfeiffer cell line, containing the heterozygous EZH2 mutation A677G,is shown to be sensitive to EZH2 inhibition by a small moleculeinhibitor Compound #13. Proliferation inhibition is seen as early as 96hr post-inhibitor treatment. The proliferation IC₅₀ after 11 days forPfeiffer cells treated with Compound #13 is 0.4 nM, compared toWSU-DLCL2 cells, which contain the Y641F heterozygous mutation, and havean IC₅₀ of 4.9 nM or OCI-Ly19 cells, which are WT for EZH2, and have anIC₅₀ of 430 nM. These results show that in a cellular context, thesensitivity of WT and mutant EZH2 to inhibition by small moleculeinhibitor is A677G>>>Y641F>>WT, as measured by proliferation.

EQUIVALENTS

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

1. A method for treating or alleviating a symptom of cancer orprecancerous condition in a subject, comprising administering to asubject expressing a mutant EZH2 comprising a mutation in the substratepocket domain as defined in SEQ ID NO: 6 a therapeutically effectiveamount of an EZH2 inhibitor.
 2. The method of claim 1, wherein themutant EZH2 is a mutant EZH2 polypeptide or a nucleic acid sequenceencoding a mutant EZH2 polypeptide.
 3. The method of claim 1, whereinthe cancer is lymphoma, leukemia or melanoma.
 4. The method of claim 3,wherein the lymphoma is selected from the group consisting ofnon-Hodgkin lymphoma, follicular lymphoma, and diffuse large B-celllymphoma.
 5. The method of claim 3, wherein the leukemia is chronicmyelogenous leukemia (CML).
 6. The method of claim 1, wherein theprecancerous condition is myelodysplastic syndromes (MDS, formerly knownas preleukemia).
 7. The method of claim 1, wherein the mutant EZH2comprises a mutation at amino acid position 677, 687, 674, 685, or 641of SEQ ID NO:
 1. 8. The method of claim 7, wherein said mutation isselected from the group consisting of a substitution of glycine (G) forthe wild type residue alanine (A) at amino acid position 677 of SEQ IDNO: 1 (A677G); a substitution of valine (V) for the wild type residuealanine (A) at amino acid position 687 of SEQ ID NO: 1 (A687V); asubstitution of methionine (M) for the wild type residue valine (V) atamino acid position 674 of SEQ ID NO: 1 (V674M); a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 685 of SEQ ID NO: 1 (R685H); a substitution of cysteine (C) forthe wild type residue arginine (R) at amino acid position 685 of SEQ IDNO: 1 (R685C); a substitution of phenylalanine (F) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F);a substitution of histidine (H) for the wild type residue tyrosine (Y)at amino acid position 641 of SEQ ID NO: 1 (Y641H); a substitution ofasparagine (N) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641N); a substitution of serine (S) forthe wild type residue tyrosine (Y) at amino acid position 641 of SEQ IDNO: 1 (Y641S); and a substitution of cysteine (C) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641C).9. The method of claim 1, wherein the EZH2 inhibitor is selected fromcompounds listed in Table
 1. 10. A method of determining aresponsiveness of a subject having a cancer or a precancerous conditionto an EZH2 inhibitor comprising a) providing a sample from the subject;and b) detecting a mutation in the EZH2 substrate pocket domain asdefined in SEQ ID NO: 6, wherein the presence of said mutation indicatesthe subject is responsive to the EZH2 inhibitor.
 11. The method of claim10, wherein the cancer is lymphoma, leukemia or melanoma.
 12. The methodof claim 11, wherein the lymphoma is selected from the group consistingof non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B-celllymphoma.
 13. The method of claim 11, wherein the leukemia is chronicmyelogenous leukemia (CML).
 14. The method of claim 10, wherein theprecancerous condition is myelodysplastic syndromes (MDS, formerly knownas preleukemia).
 15. The method of claim 10, wherein the mutation is asubstitution mutation at amino acid position 677, 687, 674, 685, or 641of SEQ ID NO:
 1. 16. The method of claim 15, wherein said mutation isselected from the group consisting of a substitution of glycine (G) forthe wild type residue alanine (A) at amino acid position 677 of SEQ IDNO: 1 (A677G); a substitution of valine (V) for the wild type residuealanine (A) at amino acid position 687 of SEQ ID NO: 1 (A687V); asubstitution of methionine (M) for the wild type residue valine (V) atamino acid position 674 of SEQ ID NO: 1 (V674M); a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 685 of SEQ ID NO: 1 (R685H); a substitution of cysteine (C) forthe wild type residue arginine (R) at amino acid position 685 of SEQ IDNO: 1 (R685C); a substitution of phenylalanine (F) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F);a substitution of histidine (H) for the wild type residue tyrosine (Y)at amino acid position 641 of SEQ ID NO: 1 (Y641H); a substitution ofasparagine (N) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641N); a substitution of serine (S) forthe wild type residue tyrosine (Y) at amino acid position 641 of SEQ IDNO: 1 (Y641S); and a substitution of cysteine (C) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641C).17. The method of claim 10, wherein the EZH2 inhibitor is selected fromcompounds listed in Table 1.